Applied Science, Faculty of
Chemical and Biological Engineering, Department of
DSpace
UBCV
Ayakar, Sonal
Doctor of Philosophy - PhD
University of British Columbia
The biomanufacturing of terpenoids is limited by the low yields of heterologously expressed biosynthetic pathway and challenges associated with recovering these products at commercial scales. To enhance the flux through methylerythritol phosphate (MEP) pathway for terpenoid biosynthesis, I screened soil metagenomes for more active and stable orthologs of the rate-limiting enzymes. I successfully identified three entirely novel, natural fusions of IspD and IspF, one of which improved production of lycopene from 235 mg/L to 275 mg/L and production of isoprene from 3.6 mg/L to 6.3 mg/L when compared to the native enzyme overexpression. A comprehensive study of the role of the linking domain revealed the higher activity of each of the catalytic domains and the absence of substrate channeling. Moreover, the non-natural fusions of E. coli enzymes catalyzing consecutive steps were constructed. One such a fusion of IspD and IspE yielded 281 mg/L of lycopene, whereas the best performing fusion of IspE and IspF only yielded 39 mg/L of lycopene. Further investigation of the sequence of this biocatalytic cascade concluded the commencement with the activity of IspE, followed IspD and IspF suggesting the reactive plasticity in MEP pathway. I probed the promiscuous nature of terpene synthases (TSs) through the systematic study of monoterpene synthases from Picea abies in vivo and in vitro. I uncovered the influence of intracellular expression and oxygen supply on the promiscuity of TSs. Computational analysis revealed the putative roles of the amino acid residues within the active sites and their evolutionary trajectory. Finally, the fermentation of engineered E. coli strains for carene and myrcene were scaled up to 1 L and a newer technique was developed for efficient product capture using a fluidized bed capture device (FBCD) using a hydrophobic resin. The device was easy to integrate into the existing bioreactor set up. It yielded 2-fold higher carene titers and 17-fold higher myrcene titers. In conclusion, the three aspects of the terpenoid biomanufacture studied in this work address some of the biggest challenges facing the industry and lay strong foundations for commercialization of terpenoid biomanufacturing processes that employ genetically engineered microorganisms.
BIOCATALYSIS AND BIOPROCESS ENGINEERING FOR TERPENOID PRODUCTION by Sonal Ayakar B. Pharm., Mumbai University, 2012 M. Tech., Institute of Chemical Technology, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2019 © Sonal Ayakar, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Biocatalysis and bioprocess engineering for terpenoid production submitted by Sonal Ayakar in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical and Biological Engineering Examining Committee: Vikramaditya Yadav, Chemical and Biological Engineering Supervisor Charles Haynes, Chemical and Biological Engineering Supervisory Committee Member Joerg Bohlmann, Botony Supervisory Committee Member Fariborz Taghipour, Chemical and Biological Engineering University Examiner Nobuhiko Tokuriki, Biochemistry and Molecular Biology University Examiner iii Abstract The biomanufacturing of terpenoids is limited by the low yields of heterologously expressed biosynthetic pathway and challenges associated with recovering these products at commercial scales. To enhance the flux through methylerythritol phosphate (MEP) pathway for terpenoid biosynthesis, I screened soil metagenomes for more active and stable orthologs of the rate-limiting enzymes. I successfully identified three entirely novel, natural fusions of IspD and IspF, one of which improved production of lycopene from 235 mg/L to 275 mg/L and production of isoprene from 3.6 mg/L to 6.3 mg/L when compared to the native enzyme overexpression. A comprehensive study of the role of the linking domain revealed the higher activity of each of the catalytic domains and the absence of substrate channeling. Moreover, the non-natural fusions of E. coli enzymes catalyzing consecutive steps were constructed. One such a fusion of IspD and IspE yielded 281 mg/L of lycopene, whereas the best performing fusion of IspE and IspF only yielded 39 mg/L of lycopene. Further investigation of the sequence of this biocatalytic cascade concluded the commencement with the activity of IspE, followed IspD and IspF suggesting the reactive plasticity in MEP pathway. I probed the promiscuous nature of terpene synthases (TSs) through the systematic study of monoterpene synthases from Picea abies in vivo and in vitro. I uncovered the influence of intracellular expression and oxygen supply on the promiscuity of TSs. Computational analysis revealed the putative roles of the amino acid residues within the active sites and their evolutionary trajectory. Finally, the fermentation of engineered E. coli strains for carene and myrcene were scaled up to 1 L and a newer technique was developed for efficient product capture using a fluidized bed capture device (FBCD) using a hydrophobic resin. The device was easy to integrate iv into the existing bioreactor set up. It yielded 2-fold higher carene titers and 17-fold higher myrcene titers. In conclusion, the three aspects of the terpenoid biomanufacture studied in this work address some of the biggest challenges facing the industry and lay strong foundations for commercialization of terpenoid biomanufacturing processes that employ genetically engineered microorganisms. v Lay Summary Terpenoids are the plant natural products having applications as pharmaceutical agents, flavors, fragrances, surfactants, fuel additives, polymer additives etc. There are various challenges in efficient production and purification of these terpenoids from plants. In this work, E. coli are genetically engineered to perform the terpenoid biosynthesis route identified from Norway spruce. Efforts of metabolic engineering in this area led to discoveries of novel biochemical pathways that operate at higher activity. Moreover, the fermentative production of the engineered E. coli provided insights into the evolution and activity of the enzymes from the plant to guide higher terpenoid production rates. An efficient method was developed for recovery of the terpenoid products from the fermentative process. This work will aid discovery of newer plant-based terpenoids that have wider applications as well as facilitate the development of their manufacturing in genetically engineered microbes. vi Preface This Ph.D. dissertation consists of seven chapters. The Ph.D. study was conducted by Sonal Ayakar under the direct supervision of Dr. Vikramaditya G. Yadav in the Department of Chemical and Biological Engineering at UBC. The literature review, experimental planning was done by Sonal Ayakar with direct inputs from Vikramaditya Yadav. Chapter 2 The metagenomic database was generated by Steven Hallam Lab members, UBC and the screen was conducted with Sandip Pawar. Construction of pSASDFI was conducted by Sonal Ayakar and Protiva Roy. Other natural fusion constructs were completed by Sonal Ayakar. Construction of non-natural fusion genes was done by Carmen Bayly. Expression of the enzyme chimera was done by Sonal Ayakar. Analysis, quantification of terpenoids and interpretation of the data for natural fusions was performed by Sonal Ayakar. The natural fusion discovery and application were presented as: S. R. Ayakar, S. V. Pawar. S. J. Hallam and V. G. Yadav, “Multiplying productivity using metagenomics: beyond canonical metabolic engineering”, Synthetic biology for natural products conference, Cancun, Mexico (2017). The patent application has been filed to protect the natural fusions. S. R. Ayakar and V. G. Yadav et al., Metabolic Engineering of E. Coli for the Biosynthesis of Cannabinoid Products, USPTO PCT/CA2018/051073, Sept. 2018 (Technology transfer to Inmed Pharmaceticals, Canada) vii Homology models, analysis and quantification of terpenoids for non-natural fusion was conducted by Sonal Ayakar and Adhithi Raghavan. The data interpretation was done by Sonal Ayakar. The patent application for non-natural fusions has been filed as: S. R. Ayakar and V. G. Yadav et al., Compositions and methods for biosynthesis of cannabinoids products in a prokaryote, International PCT Appl. No. PCT/CA2018/ 051074, March 2019 (Technology transferred to Inmed Pharmaceuticals, Canada) The entire chapter was written by Sonal Ayakar. Chapter 3 The experimental design, experimental work and data interpretation were done by Sonal Ayakar. The LC-MS analysis was done by Lina Madilao, UBC. The docked substrates were generated by Vikramaditya Yadav. The entire chapter was written by Sonal Ayakar. This work was presented at: S. R. Ayakar and V. G. Yadav, “Metagenomics yields new insights about metabolic flux through the non-mevalonate pathway”, DECHEMA Bioflavours, Frankfurt, Germany (2018). Chapter 4 The experimental design, experimental work and data interpretation were done by Sonal Ayakar. The monoterpene gene sequences and monoterpene standards were procured from Joerg Bohlmann. The docked substrates were generated by Vikramaditya Yadav. The computational modelling and figures were generated by Sonal Ayakar. The entire chapter was written by Sonal Ayakar. viii Chapter 5 The research idea was conceived by Sonal Ayakar. The initial planning of experiments for terpenoid capture were conducted by Sonal Ayakar and Azin Amiri. The fluidized bed capture device printing would not have been possible without Logan Phillips. The bioreactor run and optimization experiments were done by Azin Amiri. The data interpretation and product spectra analysis were done by Sonal Ayakar. The entire chapter was written by Sonal Ayakar. This work has now become Azin Amiri’s PhD project. Chapter 6 Following work was equally contributed by the co-authors. The application of lycopene producing strain developed and mentioned in chapter 2 was used in construction of biogenic photovoltaic cell. This work was published as: S. K. Srivastava, P. Piwek, S. R. Ayakar, A. Bonakdarpour, D. Wilkinson, V. G. Yadav, "A biogenic photovoltaic material", Small, 14, 1800729 (2018). A metric was developed to gauge efficiency of biomass valorization and is accepted for publication as: S.C. Patankar, S.R. Ayakar, V.G. Yadav, and S. Renneckar, “The V-factor: A new metric for gauging the efficiency & profitability of manufacturing processes in the bio-economy”, Bioproducts Business, 2018.bpb.047, accepted manuscript (2019). ix A chemical catalytic process was developed for lignin vaporization with highest reported yield for vanillin: S.C. Patankar, L. Liu, L. Ji, S.R. Ayakar, V.G. Yadav, and S. Renneckar, Isolation of phenolic monomers from kraft lignin using a magnetically recyclable TEMPO nanocatalyst. Green Chem. 21, 785–791 (2019). The study on characterization of microbial degradation of nanofibrillated cellulose. Manuscript on this work is under preparation. x Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ...........................................................................................................................x List of Tables ............................................................................................................................ xviii List of Figures ...............................................................................................................................xx List of Equations .................................................................................................................... xxviii List of Abbreviations ............................................................................................................... xxix Glossary ................................................................................................................................... xxxii Acknowledgments .................................................................................................................. xxxiii Dedication .................................................................................................................................xxxv Chapter 1: Introduction ................................................................................................................1 1.1 Terpenoid chemical space ............................................................................................... 1 1.2 Applications of terpenoids .............................................................................................. 2 1.3 Isoprenoid synthesis ........................................................................................................ 3 Chemical synthesis ...................................................................................................... 3 Semi-synthesis ............................................................................................................ 3 Biosynthesis in heterologous hosts ............................................................................. 4 1.4 Biosynthetic pathways of isoprenoid production ............................................................ 4 C5 precursor synthesis ................................................................................................ 4 Chain elongation ......................................................................................................... 6 xi Pyrophosphate group removal .................................................................................... 7 Scaffold activation/modification ................................................................................. 7 1.5 MEP pathway .................................................................................................................. 8 Dxs (1-deoxy-d-xylulose 5-phosphate synthase) ........................................................ 8 IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase) ................................ 9 IspD (YgbP, 4-diphosphocytidyl-2-C-methylerythritol synthase) ............................. 9 IspE (YchB, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase) .......................... 10 IspF (YgbB, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) ................... 11 IspG (GcpE, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase) ................... 12 IspH (LytB, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase) ................... 12 Idi (Isopentenyl diphosphate delta-isomerase) ......................................................... 13 1.6 Properties of isoprenoids............................................................................................... 15 Chemical properties .................................................................................................. 15 Physical properties .................................................................................................... 16 1.7 Bioprocess development for isoprene production ......................................................... 16 1.8 Challenges in terpenoid production .............................................................................. 17 1.9 Research questions ........................................................................................................ 19 1.10 Organization of thesis ................................................................................................... 21 Chapter 2: Study of enzyme fusions to improve MEP pathway yield ....................................22 2.1 Introduction ................................................................................................................... 22 Improvements in the precursor supply for the MEP pathway .................................. 22 MEP pathway optimization....................................................................................... 23 2.1.2.1 Homologous expression of MEP pathway enzymes ......................................... 23 xii 2.1.2.2 Heterologous expression of MEP pathway enzymes ........................................ 23 2.1.2.3 Improvement in genomic MEP control ............................................................. 23 2.1.2.4 Evolution of MEP pathway genes ..................................................................... 24 Effect of culture conditions on the solubility of pathway enzymes .......................... 24 Bifunctional enzyme discovery and applications ..................................................... 24 2.1.4.1 IspDF discovery and structure .......................................................................... 24 2.1.4.2 Interactions of IspD, IspE and IspF .................................................................. 25 Naturally occurring enzyme fusions catalyzing non-consecutive pathway steps ..... 27 Knowledge gap in the understanding of the MEP pathway ...................................... 27 Construction of non-natural fusions .......................................................................... 28 2.1.7.1 Demonstration of non-native function by fusions ............................................ 29 2.1.7.2 Non-natural fusions of MEP pathway enzymes ................................................ 29 2.2 Materials and methods .................................................................................................. 30 Metagenomic screening for orthologs of MEP pathway enzymes ........................... 30 Strains, genes and plasmids ...................................................................................... 31 Non-natural protein fusions ...................................................................................... 35 Construction of plasmids and strains for analysis of non-natural fusions ................ 36 Culture conditions ..................................................................................................... 38 Isoprene analysis ....................................................................................................... 38 Lycopene analysis ..................................................................................................... 38 2.3 Results ........................................................................................................................... 39 Discovery of three IspDF fusions ............................................................................. 39 Construction of MEP pathway operon ...................................................................... 44 xiii Influence of novel fusion enzymes on the MEP pathway ......................................... 46 Role of IspE .............................................................................................................. 48 Effect of linkers on IspDF1 activity .......................................................................... 49 Effect of linkers on non-natural fusions of monofunctional IspD and IspF ............. 51 Effect of XL on non-natural fusions of monofunctional E. coli IspD and IspF ....... 52 Expression of domains of IspDF1 as IspD1 and IspF1 ............................................... 52 Non-natural fusions of IspE and their effects on MEP pathway flux ....................... 53 Categorical inferences ........................................................................................... 54 2.4 Discussions ................................................................................................................... 55 Influence of native Dxs, IspD, IspF and Idi on MEP pathway flux .......................... 55 IspDF fusion activity and the influence of IspE on MEP pathway flux ................... 56 Study on linker types ................................................................................................ 57 Influence of IspDF1 domain separation on MEP pathway flux ................................ 58 Improvement in the flux on IspDE fusion ................................................................ 59 Chapter 3: Study of MEP pathway biochemistry .....................................................................61 3.1 Introduction ................................................................................................................... 61 Kinetics of MEP pathway cascade from MEP to MEcPP ........................................ 61 Canonical MEP pathway and plausible bifurcation step .......................................... 62 3.2 Materials and methods .................................................................................................. 64 Strains, plasmid and genes ........................................................................................ 64 Media and growth conditions.................................................................................... 65 Protein extraction and IspE purification ................................................................... 66 IspE in vitro reactions ............................................................................................... 66 xiv Bioluminescent assay for ATP consumption ............................................................ 67 HPLC analysis of ATP, ADP and AMP ................................................................... 67 LC-MS analysis of MEP and ME-2,4-PP ................................................................. 67 Computational analysis ............................................................................................. 68 3.3 Results ........................................................................................................................... 68 Protein expression and quantification ....................................................................... 68 Analysis of IspE reaction by ATP bioluminescent assay ......................................... 69 HPLC analysis of IspE reactions .............................................................................. 71 LC-MS analyses of IspE reaction mixture ................................................................ 74 Computational analysis ............................................................................................. 76 3.4 Discussions ................................................................................................................... 78 Chapter 4: Study of catalytic promiscuity of terpene synthases .............................................79 4.1 Introduction ................................................................................................................... 79 Catalytic promiscuity of TSs .................................................................................... 80 Factors responsible for TS promiscuity .................................................................... 81 Study of TS activity and promiscuity ....................................................................... 82 Research variables and scope .................................................................................... 85 4.2 Materials and methods .................................................................................................. 86 Strains and plasmid construction .............................................................................. 86 Media and growth conditions.................................................................................... 87 In vitro reactions ....................................................................................................... 88 Metabolite analyses ................................................................................................... 89 Computational analysis ............................................................................................. 89 xv 4.3 Results ........................................................................................................................... 91 Plasmid construction and protein expression ............................................................ 91 Study of parameters on SACar fermentations .......................................................... 93 Study of strain and fermentation parameters on monoterpene production ............... 96 Aerobic and micro-aerobic fermentations .............................................................. 104 Metabolite analyses ................................................................................................. 107 Study of terpene synthase catalytic mechanisms .................................................... 109 Cyclization of primary geranyl carbocation (cisoid) to the α-terpinyl carbocation 112 Formation of carene from the α-terpinyl carbocation ............................................. 115 Pinyl cation formation by 2,7-closure of the α-terpinyl carbocation ...................... 118 Acyclic terpenoid mechanisms from cisoid primary geranyl carbocation .......... 119 4.4 Discussions ................................................................................................................. 121 Optimization of fermentation conditions ................................................................ 121 Optimization of shake flask fermentations ............................................................. 121 Product profile characterization .............................................................................. 124 Computational analyses .......................................................................................... 126 Comparison of product profile with computational analysis .................................. 129 Chapter 5: Designing an efficient bioprocess for terpenoid recovery ...................................130 5.1 Introduction ................................................................................................................. 130 Challenges in terpenoid bioprocess development ................................................... 130 Continuous ex situ recovery (CESR) ...................................................................... 131 Drawbacks of state of the art .................................................................................. 131 The objective of the study ....................................................................................... 132 xvi 5.2 Materials and methods ................................................................................................ 132 Strains and fermentation conditions ........................................................................ 132 Bioreactor set up ..................................................................................................... 133 CESR for terpenoid ................................................................................................. 133 Resins used for terpenoid capture ........................................................................... 134 Screening resin for carene capture .......................................................................... 136 5.3 Results and discussions ............................................................................................... 137 Adsorption based carene recovery .......................................................................... 137 Bioreactor parameter optimization ......................................................................... 138 Terpenoid product analyses .................................................................................... 139 Terpenoid recovery comparison ............................................................................. 140 Chapter 6: Other scholarly contributions ...............................................................................142 6.1 A Biogenic Photovoltaic Material166 .......................................................................... 142 6.2 Isolation of phenolic monomers from kraft lignin using magnetically recyclable TEMPO nanocatalyst173 ......................................................................................................................... 145 6.3 The V-factor: Towards a new metric for gauging the efficiency & profitability of manufacturing processes for the bioeconomy ......................................................................... 149 6.4 Microbial growth and its characterization on nanocellulose synthesized from cherry veneer 153 Chapter 7: Conclusions .............................................................................................................158 7.1 Improvement in the intracellular pool of C5 precursors ............................................. 159 7.2 Understanding the biocatalytic sequence of steps in the MEP pathway ..................... 161 7.3 Study of terpene synthase biocatalysis ....................................................................... 162 xvii 7.4 Bioprocess development for terpenoid recovery ........................................................ 163 7.5 Future work ................................................................................................................. 165 Improvement in MEP pathway flux (Chapter 2) .................................................... 165 MEP pathway biochemistry (Chapter 3) ................................................................. 166 Terpene synthase study (Chapter 4) ........................................................................ 166 Terpenoid recovery from the bioprocess (Chapter 5) ............................................. 167 Bibliography ...............................................................................................................................168 Appendices ..................................................................................................................................179 Appendix A Fusion protein sequences ................................................................................... 179 A.1 IspDF1 ..................................................................................................................... 179 A.2 IspDF2 ..................................................................................................................... 179 A.3 IspDF3 ..................................................................................................................... 180 Appendix B HPLC chromatograms for ATP, ADP and AMP analysis ................................. 181 Appendix C GC-MS analysis for terpenes .............................................................................. 182 xviii List of Tables Table 1.1 Theoretical yield of MEP and MVA pathway27 ............................................................. 6 Table 1.2 Kinetic parameters of MEP pathway reactions ............................................................. 14 Table 1.3 Classes of terpenoids .................................................................................................... 15 Table 2.1 Strains, genes and plasmids used for MEP pathway study ........................................... 32 Table 2.2 Types of linkers used in the study and their sequences ................................................ 35 Table 2.3 List of non-natural protein fusions ................................................................................ 36 Table 2.4 Strains and plasmid expressing non-natural fusion proteins ........................................ 36 Table 2.5 Protein alignment analysis of the bifunctional enzymes against E. coli IspD-IspF and cjIspDF using the online BLASTN search tool ............................................................................ 40 Table 2.6 Protein alignment analysis of each domain of the bifunctional enzymes against corresponding E. coli monofunctional enzymes using the online BLASTN search tool ............. 42 Table 2.7 Protein alignment analysis of each domain of the bifunctional enzymes against corresponding cjIspDF enzyme domains using the online BLASTN search tool ........................ 43 Table 3.1 Enzyme amount and kinetics for MEP pathway steps (data extracted from118) ........... 62 Table 3.2 Strains, plasmids and genes .......................................................................................... 65 Table 3.3 Reaction conditions for different experimental sets ..................................................... 69 Table 4.1 Strains, plasmids and genes .......................................................................................... 86 Table 4.2 Summary of fermentation conditions selected ............................................................ 104 Table 4.3 Monoterpene product distribution and percent total ................................................... 108 Table 4.4 Physical properties of monoterpenes .......................................................................... 123 Table 5.1 Resins employed in the study and their properties ..................................................... 135 xix Table 5.2 Screening resin for carene recovery ............................................................................ 137 Table 5.3 Product distribution comparison of shake flask and bioreactor scale fermentations .. 140 Table 6.1 Comparison of vanillin yield derived through oxidative depolymerization of kraft lignin using Fe@MagTEMPO catalyst with values in literature .......................................................... 147 Table 6.2 Predicted global averages of fractional dollar outputs of selected sectors ................. 152 xx List of Figures Figure 1-1 Suite of isoprenoid products .......................................................................................... 2 Figure 1-2 MEP pathway ................................................................................................................ 9 Figure 1-3 Challenges in commercialization of terpenoid bioprocess .......................................... 18 Figure 2-1 Expression of the novel IspDFs as (His)6 tagged protein expressed in E. coli BL21(DE3) and their analyses on SDS/PAGE. Lanes 1 and 5: total and purified IspDF1 extract respectively, lanes 2 and 6: total and purified IspDF2 extract respectively, lanes 4 and 7: total and purified IspDF3 extract respectively, lanes 2 and 8: protein ladder. The bands corresponding with the specific protein are highlighted with red box. .............................................................................. 40 Figure 2-2 Protein sequence alignment generated from the Centre for Genomic Regulation (CRG) M-Coffee tool. IspD-IspF is the sequences for E. coli IspD and IspF. ......................................... 41 Figure 2-3 SDS/PAGE image of the soluble protein fraction of pSASDFI. Lane 1: E. coli BL21(DE3), lane 2: protein ladder, lane 3 and 4: SASDFI. The bands corresponding to protein are: Dxs (band a, 68.2 kDa), IspD (band b, 25.7 kDa), IspF (band d, 16.9 kDa) and Idi (band c, 21.2 kDa)....................................................................................................................................... 44 Figure 2-4 Influence of rate-limiting steps on MEP pathway flux. (a) Lycopene production, (b) Isoprene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ....................... 45 Figure 2-5 Influence of novel IspDF fusions on MEP pathway flux. (a) Lycopene production, (b) Isoprene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ....................... 47 xxi Figure 2-6 Homology models for the fusion proteins generated by SWISS-MODEL tool. (a) cjIspDF66, (b) IspDF1, (c) IspDF2 and (d) IspDF3. The IspD domain is in pink, the IspF domain is in blue and linker is in green. The N-terminal residue is colored black and C-terminal residue is colored orange. .............................................................................................................................. 48 Figure 2-7 Effect of IspE overexpression on lycopene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ............................................................................................................... 49 Figure 2-8 Linkers for IspDF1 and their effect on MEP pathway flux. (a) Strains overexpressing Dxs, IspDF chimeras and Idi, (b) strains overexpressing Dxs, IspDF chimeras, IspE and Idi. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ........................................................................ 50 Figure 2-9 Linkers for non-natural fusions of E. coli IspD and IspF; and their effect on MEP pathway flux. (a) Strains overexpressing Dxs, IspDF chimeras and Idi, (b) strains overexpressing Dxs, IspDF chimeras, IspE and Idi. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ........ 51 Figure 2-10 Linkers for non-natural fusions of E. coli IspD and IspF on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ........................................................................ 52 Figure 2-11 Effect of domain separation of IspDF1 on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ............................................................................... 53 xxii Figure 2-12 Non-natural fusions of IspE and their effect on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. ............................................................................... 54 Figure 2-13 Categorical comparison of lycopene production for the various linker. ................... 55 Figure 3-1 Schematic for the flow of metabolites through the MEP pathway for IspDF, IspE. (a) Canonical MEP pathway, (b)MEP pathway bifurcation. A: MEP, B: CDP-ME, C:CDP-MEP, D:MEcPP, B’: ME-2,4-PP ............................................................................................................ 63 Figure 3-2 Bifurcation in the MEP pathway with the promiscuity of IspE. The pathway highlighted with blue arrow is the canonical MEP pathway and the step highlighted by yellow arrow marks the bifurcation step from MEP. ..................................................................................................... 64 Figure 3-3 IspE expression and analysis on SDS/PAGE. Lane 1: Total soluble protein fraction of E. coli BL21(DE3), lane 2: total soluble protein fraction of SAHisIspE induced with 0.5 mM IPTG, lane 3: protein ladder, lanes 4 and 5: purified IspE. The bands corresponding to IspE are highlighted with red box. .............................................................................................................. 69 Figure 3-4 Bioluminescent assay monitoring ATP and ADP in the IspE reactions. .................... 70 Figure 3-5 HPLC analysis of set 1 reactions to study ATP consumption. ................................... 71 Figure 3-6 HPLC analysis of set 2 reactions with higher IspE concentration to study ATP consumption. ................................................................................................................................. 72 Figure 3-7 HPLC analysis of set 3 reactions when MEP is rate limiting reactant to study ATP consumption. ................................................................................................................................. 73 Figure 3-8 HPLC analysis of set 4 reactions when ADP is used as a phosphate donor. .............. 74 Figure 3-9 Mass spectra of the peak identified as ME-2,4-PP in IspE reactions. ......................... 75 Figure 3-10 The major mass fragments of ME-3,4-PP. ................................................................ 75 xxiii Figure 3-11 Canonical substrate (MEP) and alternate substrate (ME-2,4-PP) docked on IspD. .. 76 Figure 3-12 Substrate alignments in the active site for canonical MEP pathway and bifurcation pathway. Top left image: MEP and CTP in IspE, top right image: ME-2,4-PP and CTP in IspE, bottom left image: CDP-ME and ATP in IspE, bottom right image: MEP and ATP in IspE. ..... 77 Figure 3-13 (a) Canonical substrate (CDP-ME) and (b)alternate substrate (MEPP) docked on IspE........................................................................................................................................................ 78 Figure 4-1 Enzyme evolution trajectory to achieve the newer function (reproduced133) ............. 81 Figure 4-2 Protein expression and analysis on SDS/PAGE. (a) Gel image for GPPS expression, lane 1: total protein extract of E. coli BL21(DE3), lane 2: protein ladder, lane 3: total protein extract of engineered E. coli for GPPS uninduced, lane 4: total protein extract of engineered E. coli for GPPS with band corresponding GPPS (32.4 kDa) in the red box; (b) Gel image for (His)6-MTS (induction with 0.5 mM IPTG) purified by Ni-NTA column, lane 1: myrcene synthase induced, lane 2: carene synthase induced, lane 3: myrcene synthase uninduced, lane 4: carene synthase uninduced, lane 5: protein ladder, lane 6: limonene synthase uninduced, lane 7: linalool synthase uninduced, lane 8: limonene synthase induced, lane 9: linalool synthase induced. The bands corresponding with the specific protein are highlighted with red box. The bands corresponding with the specific protein are highlighted with red box. ......................................... 91 Figure 4-3 Protein expression of (His)6-GPPS and analysis on SDS/PAGE. Lane 1: protein ladder, lane 2: purified uninduced GPPS (5 ug), lane 3: purified uninduced GPPS (10 ug), lane 4: purified induced (with 0.5 mM IPTG) GPPS (5 ug), lane 5: purified induced (with 0.5 mM IPTG) GPPS (10 ug) ........................................................................................................................................... 92 Figure 4-4 Plasmid maps for MTS expression system. (a) Myrcene synthase, (b) Linalool synthase, (c) Carene synthase, (d) Limonene synthase ................................................................................ 93 xxiv Figure 4-5 Images of cultures (a) aerobic tube culture, (b) microaerobic tube culture, (c) aerobic flask culture, (d) microaerobic flask culture. ................................................................................ 94 Figure 4-6 Effect of culturing conditions on SACar. TA: aerobic tube culture, TM: microaerobic tube culture, FA: aerobic flask culture, FM: micro-aerobic flask culture. Primary Y-axis is carene titer and secondary Y-axis is normalized carene titer. The other culture conditions were: 30 °C temperature, LBY media (supplemented with the antibiotic and 2 mM MgCl2), 18 h incubation time and 10 % dodecane overlay. ................................................................................................. 95 Figure 4-7 Effect of incubation temperature on carene production. Primary Y-axis is carene titer and secondary Y-axis is normalized carene titer. The other culture conditions were: micro-aerobic flask cultures, LBY media (supplemented with the antibiotic and 2 mM MgCl2), 18 h incubation time and 10 % dodecane overlay. ................................................................................................. 96 Figure 4-8 Effect of growth media on terpene production. (a) SACar, (b) SAMyr, (c) SALin, (d) SALim+. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2, 18 h incubation time and 10 % dodecane overlay. ......................................................................................................... 98 Figure 4-9 Effect of Mg+2 concentration on terpene production. (a) SACar in LBY media, (b) SAMyr in LBY media, (c) SALin in TB media, (d) SALim+ in LBY media. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 18 h incubation time and 10 % dodecane overlay. ......................... 99 Figure 4-10 Effect of dodecane overlay on terpene production. (a) SACar in LBY media, (b) SAMyr in LBY media, (c) SALin in TB media, (d) SALim+ in LBY media. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2 and 18 h incubation time. ....................................... 101 xxv Figure 4-11 Effect of IPTG induction on terpene production. (a) SACar, (b) SAMyr and (d) SALim+ in LBY media with 10 % dodecane overlay; (c) SALin in TB media with 30 % dodecane overlay. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2 and 18 h incubation time...................................................................................................................................................... 103 Figure 4-12 Fermentative production of monoterpenes over time. (a) SACar, (b) SAMyr, (c) SALin, (d) SALim+. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Fermentation conditions are mentioned in Table 4.2. ........................................................ 106 Figure 4-13 Isomeric structures of carbocations. (a) Cisoid geranyl carbocation, (b) transoid geranyl carbocation, (c) α-terpinyl carbocation and (d) β-terpinyl carbocation. ........................ 110 Figure 4-14 Reaction mechanisms of monoterpene synthases. Monoterpene structures’ color is based on its penultimate carbocation origin. ............................................................................... 111 Figure 4-15 Changes in the orientation of primary geranyl cation on 6,1-closure to form the α-terpinyl cation. (a) Orientation of primary geranyl cation, (b) orientation of α-terpinyl cation. The TSSs are highlighted in green. .................................................................................................... 113 Figure 4-16 Role of amino acid residues in TS active site towards cisoid primary geranyl cation reactivity. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in green, the favorable interactions for 1,6-closure are highlighted in green dotted lines and unfavorable interactions are highlighted in purple dotted lines with distance measurements. ............................................................................................................................. 114 Figure 4-17 TS active site contour towards formations of carene from the α-terpinyl cation. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in purple and the α-terpinyl cation is highlighted in green.The α-terpinyl cation interactions are xxvi highlighted in purple dotted lines and carene interactions are highlighted in green dotted lines with distance measurements. ............................................................................................................... 117 Figure 4-18 TS active site contour towards formations of pinyl cation from the α-terpinyl cation. The TS is identified in the left top corner of each box. The α-terpinyl cation is highlighted in purple and the pinyl cation is highlighted in green. The α-terpinyl cation interactions are highlighted in purple dotted lines with distance measurements. ........................................................................ 118 Figure 4-19 TS active site contour towards formations of tertiary geranyl cation from cisoid primary geranyl cation. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in purple and the tertiary geranyl cation is highlighted in green.The primary geranyl cation interactions are highlighted in purple dotted lines and tertiary geranyl cation interactions are highlighted in green dotted lines with distance measurements. ........................ 120 Figure 4-20 TS active site contour and participating amino acid residues. The TS is identified in the left top corner of each box. The surface is highlighted in grey. ........................................... 128 Figure 4-21 Predictions for roles of the amino acid residues present in TS active site and structure-function correlations ................................................................................................................... 129 Figure 5-1 Schematic for construction of fluidized bed capture device (FBCD) ....................... 133 Figure 5-2 Schematic of CESR setup ......................................................................................... 134 Figure 5-3 Bioreactor parameter optimization for efficient carene production and recovery .... 138 Figure 5-4 Experimental apparatus ............................................................................................. 139 Figure 6-1 Sequential representation of whole cell bio-PV materials. (a) molecular cloning of E. coli for expression of lycopene, (b) non-covalent surface binding of TiO2 nanoparticles resulting in core@shell-like morphology and (c) deployment of biogenic PV material towards DSSC fabrication. .................................................................................................................................. 143 xxvii Figure 6-2 Electrochemical measurements of bio-PV DSSC. (a) I-V curves, (b) open-circuit photovoltage response (time dependent) and (c) cyclic voltammetry curves ............................. 144 Figure 6-3 Oxidative depolymerization of kraft lignin using the Fe@MagTEMPO catalyst .... 146 Figure 6-4 Comparison between E-factor and V-factor ............................................................. 149 Figure 6-5 AFM height profile of nanocellulose samples synthesized from cherry veneer using Fe@MagTEMPO catalyst. (a) Freshly synthesized nanocellulose sample and (b) nanocellulose sample stored at 4 °C for 10 months ........................................................................................... 156 Figure 6-6 AFM image showing assembly of microbes on nanocellulose fibrils synthesized from cherry veneer using Fe@MagTEMPO catalyst .......................................................................... 157 Figure 0-1 HPLC chromatogram for set 2 IspE in vitro reactions. The peaks for ATP (2.49 min), ADP (2.60 min) and AMP (2.72 min) are labelled. .................................................................... 181 Figure 0-2 GC-MS Chromatograms of aerobic shake flask fermentation analysis for SACar, SAMyr, SALin and SALim+. The monoterpene retention times are: pinene 6.48 min, sabinene 7.42 min, myrcene 7.86 min, carene 8.42 min, limonene 8.88 min, ocimene 9.36 min, terpinene 9.61 min, terpinolene 10.20 min, linalool 10.35 min. ................................................................. 182 xxviii List of Equations Equation 1-1 MVA pathway stoichiometry .................................................................................... 5 Equation 1-2 MEP pathway stoichiometry ..................................................................................... 5 Equation 6-1 The unabridged V-factor equation ........................................................................ 150 Equation 6-2 V-factor equation .................................................................................................. 150 xxix List of Abbreviations ADP Adenosine diphosphate AFM Atomic force microscopy AMP Adenosine monophosphate ATP Adenosine triphosphate CDP-ME 4-Diphosphocytidyl-2-C-methylerythritol CESR Continuous ex situ recovery CTP Cytidine triphosphate DHAP Dihydroxyacetone phosphate DMA Dimethylallyl alcohol DMAP Dimethylallyl monophosphate DMAPP Dimethylallyl pyrophosphate DO Dissolved oxygen DOX 1-Deoxy-d-xylulose DOXP 1-Deoxy-d-xylulose 5-phosphate Dxs 1-Deoxy-d-xylulose 5-phosphate synthase ED Entner-Doudoroff EMP Embden–Meyerhof–Parnas ESI Electrospray ionization FPP Farnesyl pyrophosphate GAP Glyceraldehyde-3-phosphate xxx GGPP Geranylgeranyl pyrophosphate GPP Geranyl pyrophosphate GRAS Generally recognized as safe HMBPP 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate ICH International Council for Harmonisation Idi Isopentenyl diphosphate delta-isomerase IPK Isopentenyl monophosphate kinase IPP Isopentenyl pyrophosphate ISO Isopentenol IspC or Dxr 1-Deoxy-d-xylulose 5-phosphate reducto-isomerase IspD or YgbP 4-Diphosphocytidyl-2-C-methylerythritol synthase IspE or YchB 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase IspF or YgbB 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase IspG or GcpE 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspH or LytB 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase LB Luria Bertani log Po/w Logarithm of partition coefficient of a compound in oil from water MD Molecular dynamic ME 2-Methyl-d-erythritol MEcPP 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate MEP 2-C-Methyl-d-erythritol-4-phosphate MTS Monoterpene synthase xxxi MVA Mevalonate/mevalonic acid MWCO Molecular weight cut off NAD Nicotinamide adenine dinucleotide NADH Reduced nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate NADPH Reduced nicotinamide adenine dinucleotide phosphate NMR Nuclear magnetic resonance OD600 Absorbance at 600 nm wavelength ORF Open reading frame PDB Protein data bank PCR Polymerase chain reaction PDB Protein data bank RBS Ribosome binding site SDS/PAGE Sodium dodecyl sulphate poly acrylamide gel electrophoresis TPP Thiamine pyrophosphate TS Terpene synthase TSS Transition state structure vvm Volume of air passed through 1 volume of liquid per minute (in bioreactor) xxxii Glossary Cn This refers to a chemical structure containing ‘n’ number of carbon atoms. For example, C10 refers to a compound containing 10 carbon atoms. Cn This refers to the carbon atom at nth position in the structure. For example, C10 refers to the carbon atom at 10th position in the structure. Error bars The error bars in the figures denote standard deviation. xxxiii Acknowledgments I express my gratitude towards my supervisor Prof. Vikramaditya G. Yadav. He continually encouraged me at every step to lead the work and gave me the independence to take up new challenges. His guidance, educational expertise, providing resources required to conduct the research and scholarly feedback gave a direction to my work. I learned many things from him and being a Biofoundry group member that will take me a long way in my academic career. I thank Prof. Charles Haynes for his support and constructive feedback at every step of my research progress and enriching my work with his enormous experience in the field. I thank Joe and Sandip who taught me ABC of molecular biology techniques and trained me in cloning experiments. Protiva and I worked together on my first independent molecular biology project of construction of MEP pathway operon and I am grateful for her guidance and support. Discovery of natural fusions would have not been possible without the support from Prof. Steven Hallam who provided us access to the genomic library and Sandip for working together in developing metagenomic screen. I thank Carmen for answering my doubts regarding cloning techniques and constructing non-natural enzyme fusions. I thank Adhithi for her willingness to take the lycopene work forward and help in analysis. I thank Lina Madilao for her expertise and help in carrying our LC-MS analysis and interpretation of its data. Prof. Joerg Bohlmann encouraged me to study terpene synthases and provided all logistic support in terms of enzymes and their products required for the work. Every discussion with him gave me a new perspective on the activity of these enzymes and I deeply acknowledge his support and his work in the field. I thank Azin for her support in investigating the bioprocessing challenges and showing a keen interest in continuing the study in detail. The work also required engineering xxxiv expertise that was provided by Logan. I thank the contributions of Vicente and Rodrigo who were the undergraduate students worked with me at different stages helping me in cloning. I thank Prof. Scott Renneckar and Saurabh who gave me opportunities to employ my expertise to tackle the challenges in the biomass valorization field. I thank Sarvesh and Prof. David Wilkinson who realized the potential of my recombinant lycopene strain as a biogenic photovoltaic material. Both these inter-field academic collaborations stimulated my thinking and widened my research approach. I also thank the industrial collaborator Inmed Pharmaceuticals for their interest in commercializing the platform strains I developed for cannabinoid biosynthesis. I appreciate inputs from their chief scientific officer Eric Hsu in the process. This experience led to the filing of two patent applications. I thank Protiva and Benson who are now working on scale-up studies of my work. My stay in UBC would have never been enjoyable without my labmates Julia, Parisa, Roza, Gaurav, Amir and Maryam apart from other members already mentioned. I would like to thank the workshop and administrate staff members of the department for the support. I appreciate all the relations I wove during my tenure at UBC, every stitch of which is unique and colorful. Lastly, there are few people in my life whom I can never thank enough. My parents have always supported me through thick and thin. Their philosophy encouraged me to pursue higher education and seek excellence. My husband Saurabh has not only encouraged me but also reflected it every day through his deeds. My younger brother Harish always kept the innocence and charm alive in me. xxxv Dedication Dedicated to my Vibrant India 1 Chapter 1: Introduction Chemical engineering and sciences have been catering to the needs of mankind. This industry is operating at a massive scale of $5.1 trillion per year of global sales1. These include petrochemicals, polymers, bulk chemicals, consumer products and specialty chemicals. Natural products (NP), their derivatives, semi-synthetic analogs and synthetic derivatives are used in sectors like pharmaceuticals, nutraceuticals, flavors, fragrances, cosmetics, energy, agrochemicals etc., and their share in the market is increasing exponentially. Some of the examples of these natural products are isoprenoids, polyketides, alkaloids, phenylpropanoids etc. 1.1 Terpenoid chemical space Isoprenoids are one of the largest and most diverse classes of natural products with more than 80000 characterized structures2. Isoprenoids are also known as terpenoids or terpenes though terpenes are regarded as the hydrocarbon moieties which on rearrangements and functionalization lead to terpenoids containing oxygen moieties. The word ‘terpene’ was named by Otto Wallach who received the Nobel prize in 1910 for his pioneering work on terpene characterization3. Figure 1-1 depicts the isoprenoid compounds and biosynthetic interlinks that are summarized from sources on the Kyoto Encyclopedia of Genes and Genomes4. The isoprenoid backbone also forms the structural basis for many other classes of natural products such as zeatin, quinone, alkaloids, polyketides etc. 2 Figure 1-1 Suite of isoprenoid products 1.2 Applications of terpenoids Terpenoids are involved in a wide variety of biological functions of growth and development in their natural producers. Terpenoids have been evolved to function as defense molecules in plants and hence are naturally tailored to bind to physiological targets. Isoprenoids have applications in pharmaceutical, nutraceutical, food, flavor and fragrance industry. Terpenes possess high calorific value due to their hydrocarbon backbone and serve as fuel. Artemisinin is used as an antimalarial drug, taxol is used as an anticancer agent. Sandalwood oil and patchouli oil are used as fragrances, isoprene is used in polymers; and myrcene, farnesene are used as fuel additives5. 3 1.3 Isoprenoid synthesis There are the following three routes for isoprenoid synthesis: Chemical synthesis Total chemical synthesis of terpenoids involves multistep catalytic processes that suffer from poor yields due to complexity in terpenoid chemical structure. Total taxol biosynthesis involves 42 steps with 0.75 % overall yield6. Isodon polycyclic diterpene synthesis from a non-natural starting material takes 12 steps involving rigorous conditions of temperature and pressure7. (-)-Lingzhiol takes 17 steps with multiple intermediary purifications from a substrate that needs to be synthesized from petrochemical resources8. Semi-synthesis Although natural hosts such as plants produce terpenoids in very low quantities, they selectively accumulate some of the stable key precursors at higher quantities. Bacteria and yeast are engineered to produce such acyclic or cyclic polyprenic intermediates in bulk which are then purified and converted to the desired isoprenoid via chemical steps. These reactions involve one or more derivatization steps of the precursor by electronegative groups like alkoxy, phenyl or halogen followed by selective cyclization reactions9. This biogenic chemical reactions10 have lower yields and are difficult to engineer for regioselectivity. These cyclization steps utilize Lewis and Brønsted acid-promoted mechanism11. Continuous flow synthesis of artemisinin, an endoperoxide ring containing diterpene, has shown to improve the yield and lower manufacturing cost12. 4 Biosynthesis in heterologous hosts Harvesting terpenoids directly from their natural producers have several consequences. Their abundance in the natural host is low requiring large biomass processing. For example, 20 kg of bark from 5 full grown Pacific Yew trees yields 1 kg of an anticancer drug, taxol13. Moreover, the terpenoids usually occur as a mixture in the host from where the desired product needs to be purified making the downstream process time intensive and cost ineffective, for example, extraction of sweet steviosides from other bitter and salty analogs in Stevia plant14. Apart from these challenges, the recovery of the desired product from the natural producer can often be inefficient such as volatility of isoprene leads to its loss from plants before its capture15. Hence, for majority of these class of products, the growing demand exceeds the supply that can be sustained by isolation from their natural sources. Hence, alternative sources of expression of the biosynthetic pathways in heterologous hosts that provide ease of engineering and processing have gained importance. Microbial fermentations with recombinant strain has shown to produce selective terpenoids at high titers and productivities16–18. Pathway engineering tools have further improved yields19–21. 1.4 Biosynthetic pathways of isoprenoid production Biosynthesis of terpenoids in the natural host is a multistep process involving a wide range of enzymes catalyzing reactions that could be summarized as follows: C5 precursor synthesis Two isoprenoid biosynthetic pathways exist that synthesize the C5 precursors, IPP and its isomer DMAPP. Archea and eukaryotes other than plants use the mevalonate-dependent (MVA) 5 pathway exclusively to convert glucose to IPP, which is subsequently isomerized to DMAPP. Plants use both the MVA and the mevalonate-independent (MEP) pathways for isoprenoid synthesis whereas MEP is dominant in prokaryotes19,22,23. There are evidences of the existence of subcellular level compartmentalization of the isoprenoid biosynthetic pathways in higher organisms like plants. The mevalonate pathway is located in the cytoplasm and MEP pathway is located in plastids. The former leads to the formation of sesquiterpenes, triterpenes, squalene, sterols and higher polyprenoids. On the other hand, the MEP pathway is responsible for the formation of the monoterpenes, diterpenes and terpenoids required for the photosynthetic machinery (carotenoids, phytol, prenyl chain of plastoquinone)24,25. 3   + 3  + 2  ⁄ →  +  + 3  + 2  + 2  Equation 1-1 MVA pathway stoichiometry  +  + 3  + 3  ⁄ →  +  + 3  + 2  + 3  Equation 1-2 MEP pathway stoichiometry Both Equation 1-1 and Equation 1-2 summarize the stoichiometry of the isoprenoid synthesis pathways from the intermediates of glycolysis. Both MVA and MEP pathways utilize the same number of ATP molecules, but MEP requires one extra NADPH equivalent per IPP synthesized. This explains why MVA pathway engineering yields higher isoprenoid yield than MEP pathway engineering26. The substrate acetyl CoA is biosynthesized from pyruvate that liberates one CO2 and generates one NADH (that generates one ATP in electron transport chain). Acetyl CoA from the EMP pathway generates 1 molar equivalent of CO2. This leads to 4 molar 6 equivalents of CO2 for MVA versus 1 molar equivalent of for MEP. The overall theoretical yield of both the pathways in E. coli aerobic fermentation is summarized27 in Table 1.1. Pathway From glucose From Glycerol MEP 1.255 Glucose  1 IPP + 2.529 CO2 2.151 Glycerol  1 IPP + 1.454 CO2 MVA 1.5 Glucose  1 IPP + 4 CO2 + 4 NADH 3 Glycerol  1 IPP + 4 CO2 + 7 NADH Table 1.1 Theoretical yield of MEP and MVA pathway27 The MVA pathway consumes 1.5 moles of glucose or 3 moles of glycerol per mole of IPP and produces excess reducing equivalents (NADH). Regeneration of these reducing equivalents and imbalances in co-factors further decrease IPP yield. Whereas, MEP utilizes 1.196 moles of glucose per 2 moles of glycerol for every mole of IPP. Overall, MEP is stoichiometrically and thermodynamically more balanced than the MVA pathway for isoprenoid biosynthesis28. MEP pathway hence holds a potential to serve as a platform to efficiently convert carbon substrate to terpenoids. Chain elongation IPP and DMAPP polymerize head to tail to form C10 GPP. Subsequent additions of IPP to the polymerized product result in longer chain carbon scaffolds viz. C15 FPP, C20 GGPP. C30 and C40 scaffolds are formed by head-to-head condensation of two FPP or GGPP respectively. Enzymes catalyzing these reactions are called prenyltransferase. Transferases attack electron-7 deficient carbocation generated by the loss of pyrophosphate from the substrate, on electron-rich double bond of IPP to form longer chain molecule. Pyrophosphate group removal These enzymes (Type I) catalyze divalent cation-mediated abstraction of a pyrophosphate group from the scaffold product of the above step. The reaction also proceeds via stabilization of carbocation by rearrangement of double bond and hydride shift. In some cases, the reactions also cascade into cyclization of the scaffold by intramolecular carbon-carbon bond formation17. Enzymes catalyzing these steps are terpene synthases (TSs) or terpene cyclases based on the function they perform. Scaffold activation/modification The hydrocarbon scaffolds are activated by various reactions like oxidation, methylation, isomerization, hydration etc. Cytochrome P450 dependent oxygenases play an important role in the modification. TSs with specialized reaction mechanisms has been evolved from large terpene synthase superfamily by diversification of their functions. In this process of evolving themselves to generate plethora compounds with diverse functionality, TS have expanded their substrate specificity and catalytic elasticity. Most terpenes that need activation by P450 enzymes, expand their chemical diversity further as P450 oxygenases are catalytically non-specific too. This has limited their use in metabolic engineering. 8 1.5 MEP pathway MEP pathway involves following catalytic steps explained in the order of their natural occurrence (Figure 1-2). Dxs (1-deoxy-d-xylulose 5-phosphate synthase) Presence of 1-deoxy-d-xylulose 5-phosphate (DOXP) as thiamine mediated enzymatic product of transketoaldolase like enzyme Dxs from pyruvate and glyceraldehyde-3-phosphate (GAP) as substrates was reported in late 20th century29,30. Dxs The initial experiments performed in E. coli31,32 successfully incorporated 1-deoxy-d-xylulose C5 chain in isoprenoid metabolites. The activity was confirmed in bacteria, yeast, fungi and plants. The reaction involves decarboxylative activation of pyruvate (Pyr) by thiamine and subsequent condensation with glyceraldehyde-3-phosphate (GAP) and detachment to form 1-deoxy-d-xylulose 5-phosphate (DOXP)33. The 1-deoxy-d-xylulose C5 chain was also shown to be the precursor for the heterocycles of thiamine and pyridoxal34. Dxs activity controls the overall flux through the MEP pathway by acting as a gatekeeper in most of the species35. Dxs contains TPP binding site that is shown to be competitively inhibited by IPP and DMAPP in Populus trichocarpa and Populus alba36,37. E. coli Dxs bears similarity with Dxs from Populus trichocarpa and same feedback inhibition is likely to occur in E. coli. 9 Figure 1-2 MEP pathway IspC (Dxr, 1-deoxy-d-xylulose 5-phosphate reductoisomerase) Conversion of DOXP to 2-C-methyl-d-erythritol-4-phosphate (MEP) is a two-step reaction catalyzed by IspC. It involves an acid catalyzed α-cetol rearrangement reaction38 to 2-C-methyl-d-erythrose-4-phosphate followed by NADPH mediated reduction to yield MEP39 requiring Mn+2 metal ion . Presence of 5-phosphate is required for activity and 1-deoxy-d-xylulose (DOX) is not converted to 2-methyl-d-erythritol (ME)31. The exact catalytic mechanism is unknown. IspD (YgbP, 4-diphosphocytidyl-2-C-methylerythritol synthase) Reactions conducted to characterize the IspD activity were carried in E. coli cell extracts and radiolabeled MEP and cytidine triphosphate (CTP) as substrates and analyzed by NMR +DxsCO2IspCNADP+NADPH/H+PPiCTPIspDPyr GAP DXP MEPCDP-MEATPADPIspECDP-MEPCMPIspFIspGMEcPPHMBPPDMAPPIPPIdiIspH10 spectroscopy on purification40. The product was thus characterized as 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and enzymatic activity requires divalent cation like Mg+2, Mn+2 or Co+2 as a cofactor. It is the energy-intensive step in the MEP pathway. A homologous gene is also present in bacteria, eubacteria, yeast and plants. The ispF gene is present upstream ispD gene and both are cotranscribed. IspD exists as a homodimer (26 kDa as a monomer and 50 kDa as a dimer) verified from SDS/PAGE in nondenaturing conditions40. N-terminal sequencing revealed removal of the start methionine on posttranslational modification. The specific activity is reported to be 23 µmol mg-1 min-1 with Km value of 3.14 µM for MEP and 131 µM for CTP. Ribitol-5-phosphate and erythritol-4-phosphate tested negative for substrates and divalent cations Cu+2, Ni+2, Ca+2, Fe+2 and Zn+2 tested negative for cofactors. Utilization of nucleotide 5-triphosphates by wild type IspD as is CTP=100 %, UTP=30 %, GTP=20 %, ATP=20 %, ITP=17 % suggesting the enzymatic mechanism is selective to CTP40. IspE (YchB, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase) The gene ispE was discovered by systematic computer search following dxs, ispC, ispD, and ispF distribution in E. coli chromosome41. It translates to a 31 kDa polypeptide IspE that catalyzes ATP- dependent phosphorylation of CDP-ME. This was confirmed by reaction of radiolabeled CDP-ME with ATP using purified recombinant IspE41. 1H, 13C and 31P NMR spectroscopy studies of the reaction mixture and product confirmed 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP) as the major product and ADP as a by-product. NMR spectroscopy results nullified the possibility of erythritol-1-phosphate and of erythritol-3-phosphate41. Initial studies of isopentenyl monophosphate kinase (IPK), that belongs to the same group of the kinase as IpsE, from Mentha piperita and E. coli reported the promiscuous nature of 11 IspE. It converts isopentenyl monophosphate (IP) to isopentenyl diphosphate and was reported as the terminal step in MEP pathway42. It contains a plastidial targeting sequence and forms 33 kDa (M. piperita) and 31 kDa (E. coli) cytosolic polypeptide. It also exhibits activity for isopentenol (ISO) and dimethylallyl alcohol (DMA) to respective monophosphate. Dimethylallyl monophosphate (DMAP) does not serve as a substrate. Radiolabeled substrate feeding studies showed that the ease of conversion of metabolites to terpenoids is in the order IPP > ISO > IP and; DMAP and DMAPP did not get incorporated in terpenoids in vivo42. IspF (YgbB, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) The recombinant protein was found to convert CDP-MEP into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) and CMP using Mn+2 or Mg+2 as a cofactor. It also converts CDP-ME to in vitro product 2-C-methyl-D-erythritol-3,4-cyclophosphate but is not involved in isoprenoid biosynthesis43. Presence of the reaction cascade comprising the reactions catalyzed by Dxs, IspC, IspD, IspE and IspF was also confirmed in C. annuum chloroplasts. The crystal structure of the enzyme revealed the presence of an extra electron dense cavity that can bind to several isoprenoid diphosphates (like IPP, DMAPP, GPP and FPP) with GPP exhibiting the best fit. The Arg 142 δ-guanido side chain in the cavity interacts with the diphosphate and hydrophobic part of the cavity interacts with a hydrocarbon chain of GPP molecule44. But these interactions are not reported as catalytic. In vitro studies could not confirm feedback regulation by the prenyl diphosphates, but showed that IspF exhibits enhanced activity in the presence of MEP and ME. This effect was diminished by FPP45. 12 IspG (GcpE, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase) IspG catalyzes NADP mediated reductive rearrangement of MEcPP into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP) catalyzed by cysteine residues. IspG in E. coli has three conserved cysteines residues (269, 272, and 305) that is postulated to form a disulfide bond participate in the reaction via reductive cleavage if disulfide bond by an attack from electronegative C2 center of the substrate and the product was verified by radiolabeling assay46. Another study found that oxygen-sensitive Fe/S redox cluster is necessary for IspG apoenzyme activity and is present as a [4Fe-4S] protein47. ErpA protein is essential for the assembly of the [4Fe-4S] cluster in IspG48. The association of the prosthetic group with IspG was verified by UV/Vis spectroscopy. These studies were performed with cell-free extracts as well as with purified enzyme in the presence of flavodoxin, flavodoxin reductase and NADPH as regeneration system. The substrate MEcPP accumulates in E. coli under oxidative and nitric oxide stress due to the dependence of IspG on radical oxygen species. HMBPP is also reported to be toxic to IspG activity36. IspH (LytB, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase) Gene ispH was found to be essential for viability of the cell and E. coli with ispH deletion only survived in media supplemented with mevalonate suggesting its role in MEP pathway49. Both IspG and IspH reactions involve the transfer of 2 electron equivalents and C-OH bond cleavage involving Fe-S cluster as prosthetic group50. It catalyzes the conversion of HMBPP to IPP as well as DMAPP. The catalytic site contour determines the ratio of IPP to DMAPP. It is 5:1 in E. coli46,51, 15:1 in Ginko biloba52 and 2:1 in Burkholderia glumae53. The point mutation G120A in IspH lead to DMAPP as the sole product54. 13 Idi (Isopentenyl diphosphate delta-isomerase) The gene was first isolated from Saccharomyces cerevisiae55. Idi catalyzes the terminal step of both the mevalonate and MEP pathway. The reaction involves the conversion of the homoallylic IPP to its highly electrophilic isomer dimethylallyl diphosphate (DMAPP). The mechanism involves 1,3-allylic rearrangement mediated by Cys67, Glu116 and Tyr104 residues in E. coli56,57. Idi is a 20 kDa protein and is present in bacteria, yeast and eukaryotes55. This terminal step adjusts the distribution of IPP and DMAPP but is not essential for E. coli as IspG can produce IPP and DMAPP unlike MVA pathway52. Table 1.2 summarizes the rates and kinetic parameters of the enzymes in the pathway. . 14 Table 1.2 Kinetic parameters of MEP pathway reactions Enzyme Maximum rate (µM.min-1. mgenz-1) Km (µM) Kcat (s-1) Reaction conditions Ref. Dxs - 23.5 (GAP), 48.7 (Pyr) 153.6 100 mM HEPES, 2 mM MgCl2, 2.5 mM tris(2-carboxyethyl)phosphine, 100 µM NADPH, 1 mM thiamine diphosphate as cofactor 58 IspC 35 45 (DXP), 0.5 (NADPH) 29 50 mM HEPES at pH 7.6 and 3 mM MgCl2, 150 µM NADPH 59 IspD 23 3.14 (MEP), 131 (CTP) - 100mM Tris-HCl pH 8.0, 20 mM sodium fluoride, 10 mM MgCl2 40 IspE 34 200 (CDP-ME), 20 (ATP) - 5 mM MgCl2, 60 mM NaCl, 20 mM HEPES, 1 mM dithiothreitol (DTT), 0.5% DMSO, 200 µM CDP-ME (Echelon), and 40 µM ATP 60,61 IspF - 410 (CDP-MEP) 2.7 1.2 mM NADH, 6 mM MgCl2, 18 mM phosphoenolpyruvate, 6 mM ATP, pyruvate kinase (3 U), CMP kinase (1.5 U) 62 IspG [4Fe-4S]+ 0.55 311 (MEcPP) 0.4 [Fe]:[S]:[IspG]=3.9:4.3:1, 1.0 mM reduced methyl viologen in 100 mM Tris-HCl, pH 8.0 63 IspH 30.4 31.6 (HMBPP) 1125 100 mM Tris-HCl pH 8.0, 1.0 mM HMBPP, 50 nM IspH, 5 mM dithionite, and 1 mM 6,7-dihydro-2,11-dimethyldipyrido[1,2-a:2,1-c]pyrazinium dibromide as mediator 64 Idi 4345 9.5 - 50 mM Tris-HCl buffer, pH 7.4, containing the divalent metal cofactors (1.5 mm Mn2+ and Mg2+) and [1-14C]IPP 18 μm 56 15 1.6 Properties of isoprenoids Chemical properties The diversity in the chemical structure of isoprenoids is the outcome of multiplicity and order of a few basic plug-and-play reactions. Terpenes are labeled based on the number of C5 units they contain (Table 1.3). These units are joined in ‘head to tail’ manner except squalene and carotenoids that are joined in tail to tail manner with a basic molecular formula of (C5H8)n where n is a number of monomeric units. Table 1.3 Classes of terpenoids Terpenoid class Number of isoprene units involved in the biosynthesis Number of carbons Hemiterpene 1 5 Monoterpene 2 10 Sesquiterpene 3 15 Diterpene 4 20 Sesterterpene 5 25 Triterpene 6 30 Tetraterpene 8 40 Polyterpene n (where, n>8) 5n Terpenoids are acyclic or cyclic and are non-aromatic. The geminal alkyl groups in the polyprenyl intermediates limit to cyclization to 3, 4 or 6-membered rings. This effect is known as ‘gem dialkyl rule’ postulated by Ingold in 1921. Terpenoids also contain oxygen atoms that are 16 generally present as hydroxyl groups (linalool, phytol), ether (cineole), ketone (verbenone) and endoperoxide (artemisinin) among others. Terpenes belonging to the same class are structural isomers of each other. Terpenoids also have chiral centers and exhibit stereoisomerism (enantiomers around chiral carbon). They yield isoprene as a major product on thermal degradation. Terpenoids are also prone to oxidation and addition reactions due to the presence of unsaturation. Physical properties Most terpenoids are colorless fragrant liquids (exceptions like camphor that is solid at room temperature) with densities lower than water and are volatile in nature. Terpenes have very close boiling points and are difficult to separate from a mixture by distillation. Terpenes are hydrophobic compounds with high log Po/w values. Terpenoids have oxygen moieties that impart polarity to the molecule but are not completely ionized. Terpenoids are water immiscible and are extracted in organic solvents. 1.7 Bioprocess development for isoprene production The biochemical properties of terpenoids make them excellent candidates for an efficient bioprocess. They are secondary metabolites and are commonly secreted out of the cell. Despite many of their economically interesting applications, there occur very few examples of their commercial production. Because of their cytotoxicity and volatility, the complete bioprocess must include in situ product recovery. 17 1.8 Challenges in terpenoid production The commercial production of terpenoids using recombinant strain encounters several challenges. The yield of heterologously expressed terpenoid biosynthesis is low. This challenge can be broken down into two parts. The first factor that is responsible for the overall yield of the biosynthesis is the MEP pathway. And, the flux through the MEP pathway is very low resulting in a lower abundance of intracellular IPP and DMAPP pool. The second factor is that the downstream step catalyzed by terpene synthases results in a generation on various side products due to their catalytic promiscuity. When these factors were taken care of resulting in the development of a recombinant selectively making the desired terpenoid at high yield, the recovery of the product is challenging. The current state-of-the-art of biphasic extraction is not scalable. These challenges are summarized and represented in Figure 1-3. 18 Figure 1-3 Challenges in commercialization of terpenoid bioprocess 19 1.9 Research questions The flux through heterologously expressed plant secondary metabolic pathways is typically quite low. Metabolic engineering, a branch of synthetic biology, is used to modulate the fluxes through these pathways through overexpression of rate-determining enzymes in the pathway. Such a robust and optimized precursor platform can be used to study isoprenoid biosynthesis with high throughput. This chassis can be used to mine new terpenoids65 that would otherwise remain undetected in the native hosts due to lower abundance. As a consequence, the key questions considered in this thesis include: • Can more active and stable orthologs of MEP pathway enzymes improve terpenoid production? Protein engineering has been employed to improve enzyme throughput. On the other hand, a third strategy that involves replacing bottleneck steps with more active and/or stable orthologous enzymes has not witnessed widespread adoption. The fusion enzymes have shown to improve the efficiency of biocatalysis through substrate channeling and decreasing diffusional limitations. The study was conducted to mine such fusions from the soil metagenome for MEP pathway enzymes. • What are the effects of different linkers on the fusions of enzymes involved in the MEP pathway? The IspDF bifunctional enzymes have shown to improve flux through the MEP pathway due to the co-localization of active centers. Effect of linkers on the localization and the flux 20 is not studied. Linker flexibility/rigidity will play a role in the in situ association of the enzymes (IspD, IspE and IspF) forming a catalytic complex66. • Why natural fusions of IspD and IspF occur but not the fusions of IspE? IspD and IspF catalyze non-consecutive steps in the MEP pathway and their fusions are widely found in proteobacteria. Fusions of IspE are not reported that catalyzed the intermediary step. Study of non-natural fusions of IspE and various fusion variants of IspDF can provide insights about MEP pathway control and engineering. • What factors influence heterologously expressed TS promiscuity? TSs are reported to be one of the highly promiscuous classes of enzymes. Various amino acid sequences at the active site control the catalytic mechanism and selectivity. The distinction between the in vitro and in vivo catalytic promiscuity of these enzymes is unclear. I planned to undertake a systematic study of this heterologous system. • How terpenoids can be efficiently recovered from the bioreactor? The current strategy of terpenoid recovery from the bioreactor involves the overlay of an organic solvent suffers from many disadvantages. First that they are high boiling solvents and isolation of terpenoids is energy and cost ineffective. Developing proof of concept of utilization of resins for de novo terpenoid fermentation is established through this study. 21 1.10 Organization of thesis Chapters 2 2 3 4 5Sections  MEP flux improvement Study of non-natural enzyme fusionsStudy of MEP pathway biochemistryStudy of terpene synthase promiscuityDevelopment of efficient terpenoid bioprocessDescription  Discovery of three novel IspDF bifunctional enzymes.Analysis of linkers. Construction of non-natural fusions of IspDF1domains and E. coli IspD, IspF, IspE.In vitro reactions of IspE and; in silico analysis of IspE, IspD and IspF.Expression of TSs and analysis of products.Considerations to the efficient product recovery.Output  IspDF1 at least 17% more active enzyme than native homologs.Higher activity of each of the domains of IspDF1. 2.3-fold improvement in the lycopene yield with IspDE fusion.Detection of a diphosphate intermediate and discovery of a branch point within MEP pathway.Evidence of influence of cellular environment on catalytic intermediates. Involvement of specific amino acid residues to catalytic function.Design of adsorption based ex situ continuous system for terpenoid recovery.Deliverables  Patent 1 filed. Patent 2 in preparation. Manuscript 1 ready for submission. Manuscript 2 in preparation.Manuscript 3 in preparation.22 Chapter 2: Study of enzyme fusions to improve MEP pathway yield 2.1 Introduction Flux through the MEP pathway in E. coli is very low but the pathway is necessary for cell viability. The disruption of the pathway genes was reported to be lethal in E. coli67,68. The pathway downstream to Dxs catalytic step can be complemented with heterologous expression of the rate determining enzymes of the MVA pathway69. Dxs deletion cannot be complemented with MVA pathway because of its role in vitamin B6 and B1 biosynthesis34. Whereas IPP and DMAPP are essential for prenylation of t-RNAs70 and quinones71. As discussed in section 1.3 (page number 4), MEP operates at a higher theoretical yield and is thermodynamically favored over MVA pathway27. The experimentally observed MEP pathway yield is far from the theoretical maxima. MEP pathway can be used to generate a most robust heterologous platform for isoprenoid biosynthesis on optimization. Improvements in the precursor supply for the MEP pathway GAP and pyruvate are the metabolites from the glycolytic pathway involved in central carbon metabolism. Efforts of improving flux through glycolysis have been limited by the attempts at enhancing sugar uptake rate72–74. As the glucose transporter was made more active, various steps in the glycolytic pathway lost their metabolic control75. The thermodynamics of conversion of fructose-1,6-diphosphate to DHAP and GAP push the equilibrium towards the substrate76. Isomerization of DHAP and GAP is favored towards DHAP. Some successful efforts have been to channel the flux through the pentose phosphate pathway and ED pathway for isopentenol production77. The distribution between GAP and pyruvate has a role in driving flux through the 23 MEP pathway and redirection of flux to GAP from pyruvate lead to improvement in downstream lycopene production78. The same study also reported that feeding GAP and pyruvate does not change the flux substantially. MEP pathway optimization Improvements in genome sequencing, genome mining, proteomics, metabolomics and bioinformatic tools have provided the field of metabolic engineering to find wider applications. 2.1.2.1 Homologous expression of MEP pathway enzymes A well-studied strategy is pathway optimization through tools of metabolic engineering. Overexpressing homologous MEP pathway bottlenecks have proven to greatly enhance the synthesis of terminal isoprenoid products. Overexpression of four genes- dxs, ispD, ispF and idi were shown to improve taxol yield in E. coli28. Whereas, overexpression of dxs, ispD, ispF and ispH improved lycopene yield by 15-fold in Bacillus subtilis79. 2.1.2.2 Heterologous expression of MEP pathway enzymes MEP flux can be upregulated by expression of higher active heterologous MEP pathway enzymes. This involves the replacement of a single enzyme or the entire pathway construct with orthologous enzymes. For example, Dxs from Arabidopsis thaliana was expressed in transgenic Lavandula latifolia led to a 5-fold higher total terpenoid yield80. 2.1.2.3 Improvement in genomic MEP control The genes involved in the MEP pathway are controlled by constitutive promoters. Chromosomal exchange of dxs promoter with a strong promoter Ptuf in Corynebacterium glutamicum achieved 60 % improved Dxs activity and doubled lycopene production51. 24 2.1.2.4 Evolution of MEP pathway genes Reasons for flux limitations lie in one or more of these factors: low activity, low stability, low expression levels, low solubility, feedback regulation or toxicity. The approach of modification of these enzymes at genetic levels through mutation has been tried. Directed co-evolution of Dxs, Dxr and Idi lead to 60% improvement in lycopene yield in E. coli81. Effect of culture conditions on the solubility of pathway enzymes Dxs, IspG, IspH and Idi suffer from low solubility and form inactive inclusion bodies on overexpression. Improvement in the solubility hence will greatly impact their activity. Lowering incubation temperature, co-expression with chaperone proteins and protein mutagenesis improve the solubility of the otherwise insoluble protein. Another strategy of supplementing growth media with betaine and sorbitol increased the Dxs solubility by 60%. This also led to overall improvement in the MEP pathway flux82. Bifunctional enzyme discovery and applications The occurrence of fused IspDF enzyme is common in α and ε proteobacterial genomes but not so in β and γ proteobacterial genomes83. IspDF is isolated and studied in detail from Campylobacter jejuni83, Mesorhizobium loti84 and Agrobacterium tumefaciens85. 2.1.4.1 IspDF discovery and structure The first bifunctional gene was isolated from Campylobacter jejuni83, a product of which (cjIspDF, 42 kDa polypeptide) catalyzed two reactions individually carried out by IspD and IspF with rates of 3.9 µmol.mg-1.min-1 and 0.8 µmol.mg-1.min-1 respectively. These rates are still lower 25 than that of E. coli monofunctional enzymes (Table 1.2). The cjIspDF had a greater similarity with E. coli IspF (approx. 48 %) than ispD (approx. 25 %). In vitro reactions with purified His tagged protein from recombinant E. coli employing 13C labeled MEP yielded CDP-ME and addition of Zn+2 ion as cofactor gave highest rate (18.5 µmol.mg-1.min-1) with Km values of 3 µM and 20 µM for CTP and MEP respectively at pH 5. Presence of ATP did not alter the reaction kinetics until IspE was added when it led to the formation of MEcPP with the highest activity at pH 8 and Ca+2 as a cofactor with 19 µM Km value for CDP-MEP. The estimated shortest distance between the two catalytic centers of IspD and IspF subunits in the cjIspDF is around 38 Å. The cjIspDF was reported to exist as a trimer, hexamer and dodecamer when analyzed by size exclusion chromatography83 whereas, the crystal structure is hexameric66. It also shows two clear domains for each of the domains joined by a linker sequence. The hexameric assembly contains two trimers of IspD domain dimers and two trimers of IspF domain trimers. In this hexameric complex, one of the IspF domains of corresponding dimers IspD domains associate to form trimers. This means that the individual domains of the same bifunctional polypeptide do not associate. Another well studied bifunctional IspDF from Mesorhizobium loti (mlIspDF) was expressed in E. coli and was also found to exhibit catalytic activities of both IspD and IspF84. The IspD subunit had 46 % similarity with E. coli IspD whereas, The IspF subunit had 44 % similarity with E. coli IspF. Size exclusion chromatography of the protein sample showed the existence of monomeric unit and dimeric complex of mlIspDF. Higher molecular complexes were not observed. 2.1.4.2 Interactions of IspD, IspE and IspF Experiments on monomeric E. coli enzymes were performed and analyzed by sedimentation velocity method for 3 sets of combinations: (a) IspD and IspE, (b) IspE and IspF; 26 and (c) IspD, IspE and IspF. These studies revealed the assembly of three IspD dimers, three IspE dimers, and two IspF trimers66. The same study revealed that the domains IspD and IspF from IspDF associate with IspE to form a mega complex66 and aid the substrate channeling. This was reported for both cjIspDF and atIspDF85 (IspDF from Agrobacterium tumefaciens) Trimers of IspD dimer and IspE dimer complex with dimers of IspF trimers to form an assembly of 18 catalytic centers. atIspDF was also detected to associate at higher molecular weight ratios. For cjIspDF, the distance between the two catalytic centers of the same multimer is 35 Å for IspD subunit and 30 Å for IspF subunit which is lesser than the distance between the two catalytic centers of the cjIspDF. On the other hand, a similar study85 was done on IspDF and IspE isolated from Agrobacterium tumefaciens (atIspDF and atIspE respectively). These enzymes were not found to associate based on sedimentation velocity experiments. Further validation was confirmed in vitro condition by adding an inactive form of atIspE by A152A point mutation. The inactive IspE did not change the reaction course of conversion of MEP to MEcPP through atIspDF and atIspE cascade. The mutated IspE should have interacted with the complex and lowered the overall rate of reaction if the enzymes associate to facilitate substrate channeling. The other examples of fusions where the active sites but do not channel the substrates. GlmU enzyme from E. coli involved in peptidoglycan biosynthesis is a bifunctional enzyme that catalyzes the consecutive steps in the pathway but the intermediate is released from the first active site, accumulates in the environment to be acted upon by the second functionality86. 27 Naturally occurring enzyme fusions catalyzing non-consecutive pathway steps Natural occurrence of fusions enzymes that catalyze non-consecutive steps in a biosynthetic pathway is rare25. Gram-positive bacteria like Enterococcus faecalis and Enterococcus faecium encode a bifunctional enzyme MvaE that possesses both 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities that are involved in MVA pathway and are separated by one step catalyzed by HMG-CoA synthase87,88. But no association complex is reported. The second example is involved in the carotenoid biosynthetic pathway. The carRA gene identified in fungi - Phycomyces blakesleeanus and Mucor circinelloides that encodes fusion for phytoene synthase and lycopene cyclase89,90. Phytoene synthase is a prenyl transferase that catalyzes the synthesis of phytoene from the condensation of two GGPP molecules. Phytoene is then converted into lycopene by the dehydrogenase encoded by CarB. β- Carotene is then synthesized by cyclization catalyzed by lycopene cyclase. The reports accept the presence of exceptions of these fusions, they fail to justify the reason as well as usefulness. The occurrence of enzyme fusions at the genetic level is common. Fatty acid synthesis, polyketide synthesis pathways involve bifunctional enzymes but all of them catalyze consecutive steps in the pathway. The reasons behind the existence of the fusions like IspDF, MvaE and CraAR remain unclear. Though some researchers argue their relevance at metabolic control levels. Knowledge gap in the understanding of the MEP pathway There lies a gap between theoretical maximum and experimentally feasible yield of the MEP pathway. Many efforts are done in the area of genome engineering, protein engineering and metabolic engineering to fill in the gap. A strategy that involves replacing the bottleneck steps 28 with more active and/or stable orthologous enzymes has not witnessed widespread adoption. The bifunctional enzymes that are reported to be involved in the pathway are promising targets. There are no reports of influence on in vivo MEP flux by these bifunctional IspDFs. The efforts have been directed towards studying the purified proteins for their in vitro activities. In this work I conducted metagenomic screening for identification of fusions of enzymes of the MEP pathway with consideration to enhance substrate channeling. All the fusions discovered were of IspD and IspF. These enzymes are reported to catalyze non-consecutive steps in the MEP pathway. I conducted a thorough study on the linker characteristics and their influence on MEP pathway flux. The linker sequence that connects the two domains in a bifunctional enzyme plays critical role91,92. The flexibility and rigidity of the linker play a role in maintaining independence in the movements of the domains. I non-naturally fused IspE to each of IspD and IspF to mimic natural fusions. Isoprene and lycopene were used as reporter molecules and the comparative study was conducted. Such a robust and high yielding MEP pathway platform strain can thus be utilized to produce isoprenoids as well as to mine new compounds. Construction of non-natural fusions Synthetic fusion proteins that have more than one catalytic activity are designed either to expand the catalytic spectra of the protein or to improve the catalytic efficiency. Expressing a single fusion protein also substantially reduce production cost leading to higher industrial applicability93. Chemical catalysis has widely accepted the strategy of multifunctional catalyst that is tailored to catalyze more than one type of reactions and has gained popularity in the industry94,95. There are two major ways of generating non-natural fusions96. First is at the genetic level by replacing transcriptional stop codon of the first gene and transcriptional start codon of the 29 second gene with a nucleotide sequence that will generate a peptide bond on translation. The second is introducing tags in the protein that trigger an association reaction forming the peptide bond at the post-translational step. 2.1.7.1 Demonstration of non-native function by fusions Conversion of L-erythrulose from 2-amino-1,2,3-butanetriol was catalyzed by a novel enzyme, ω-transaminase using serine as amine donor. This reaction generated hydroxypyruvate as a byproduct that was shuttled back into a substrate re-generating system as an amine donor by the action of a transketolase enzyme for the conversion of glycoaldehyde to L-erythrulose. The fusion of transaminase and transketolase created an efficient closed loop system97. Another study combined four heterologously expressed enzymes to create a multienzyme reaction cascade in E. coli for the conversion of ethylbenzenes to enantiopure (R)‐1‐phenylethanamines eliminating the need for use of additional co-factors98. 2.1.7.2 Non-natural fusions of MEP pathway enzymes There are no reports on non-natural MEP pathway enzyme fusions. Absence and presence of fusions to aid active site colocalization and thereby channeling substrate for efficient conversion are highly debated topics in the field. Moreover, the fusions of IspD and IspF occur that catalyze non-consecutive steps in the pathway and fusions of IspE have been never reported. This study has two objectives. First was to mine metagenomes for open reading frame coding for the fusion enzymes involved in the MEP pathway and; the second was to design a strategy to study the role of linker types and their influence on the pathway. 30 2.2 Materials and methods Metagenomic screening for orthologs of MEP pathway enzymes Soil samples were collected at the Skulow Lake site (SBS-3 WL) located at coordinates 52° 20’N, 121°55’W as a part of Long-term Soil Productivity (LTSP) study99. High molecular weight genomic DNA was extracted and purified to create large insert fosmid libraries100–102. NR fosmid library was created using the CopyControl™ Fosmid Library Production Kit (Epicentre) according to the manufacturer's protocol from Bt soil horizon in a naturally disturbed reference site. Twenty 384-plates from the library were Sanger end-sequenced at the Michael Smith Genome Science Center (GSC), UBC with the pCC1-Forward (5’-GGATGTGCTGCAAGGCGATTAAGTTGG) and pCC1-Reverse (5’-CTCGTATGTTGTGTGGAATTGTGAGC) primers generating ~7680 paired-end sequences. Approximately 530 fosmids were selected in silico based on phylogenetic gene markers located on the fosmid ends and functional screens and have been full-length sequenced on the Illumina HiSeq platform at the GSC. Sequence analysis including open reading frame (ORF) prediction and annotation was performed using the MetaPathways pipeline v2.5 supplied with a collection of reference databases (KEGG 2011-06-18, COG 2013-12-27, RefSeq 2014-01-18 and MetaCyc 2011-07-03)99. Protein family searches using the online HMMER tool version 2.17.3103 were performed to confirm functional annotations generated by the MetaPathways tool. The resulting MetaPathways outputs for the fosmid ends and fully sequenced fosmids were searched for Enzyme Commission (EC) numbers of genes encoding bifunctional ispDF. Cognate nucleotide sequences were searched against NCBI database using the online BLASTN search tool and resulting text files were uploaded into Megan 6.10.0 to assign taxonomy using the LCA algorithm99. Based on this 31 analysis fosmid sequences of NR0032_N05, NR0032_O07 and NR0037_N05 were assigned to Acidobacteria and the ispDFs were annotated as ispDF1, ispDF2 and ispDF3 respectively. Strains, genes and plasmids All strains, plasmids and genes used in this study are listed in Table 2.1. It contains genetic operons with natural monomeric enzymes as well as natural fusion enzymes of the MEP pathway. Genes dxs, ispD, ispE, ispF, idi were amplified from E. coli strain K12 genome by polymerase chain reaction. Bifunctional genes ispDF1, ispDF2 and ispDF3; and ispS were codon optimized and synthesized from Genewiz Inc. pTrc-trGPPS(CO)-LS was a gift from Jay Keasling (Addgene plasmid 50603)104 from where the vector backbone was amplified to construct the plasmid variants. E. coli DH5α was used as cloning host and E. coli BL21(DE3) was used as an expression host. Oligonucleotide primers were synthesized from Integrated DNA Technologies, USA. Genes were amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific). Recombinant plasmid DNA was purified using the GeneJET plasmid miniprep kit (Thermo Scientific). DNA extractions from agarose gel and PCR reactions were performed using the GeneJET gel extraction and DNA cleanup micro kit (Thermo Scientific). Wizard Genomic DNA purification kit was procured from Promega. All the restriction enzymes, DNA ladders were from New England Biolabs. Protein ladder, and AnyKD Mini-PROTEAN TGX precast protein gels were from Bio-Rad. Antibiotics, nutrient media and other reagents were purchased from Sigma-Aldrich. Protein expression was verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) on cell lysate samples. 32 Table 2.1 Strains, genes and plasmids used for MEP pathway study Strains Description Source E. coli DH5α Cloning strain NEB (# C2987) E. coli BL21(DE3) Expression strain NEB (#C2527) E. coli strain K12 Gene amplification Sigma-Aldrich (#EC1) SASDFI pSASDFI and expressed in E. coli BL21(DE3) This study SAIso pSAIspS expressed in E. coli BL21(DE3) This study SAIso-SDFI pSASDFI and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-SDF1I pSASDF1I and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-SDF2I pSASDF2I and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-SDF3I pSASDF3I and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-SI pSASI and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-DF1 pSADF1 and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-DF2 pSADF2 and pSAIspS coexpressed in E. coli BL21(DE3) This study SAIso-DF3 pSADF3 and pSAIspS coexpressed in E. coli BL21(DE3) This study SALyc pAC-LYC expressed in E. coli BL21(DE3) This study SALyc-SDFI pSASDFI and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-SDFEI pSASDFEI and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-SDF1I pSASDF1I and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-SDF1EI pSASDF1EI and pAC-LYC coexpressed in E. coli BL21(DE3) This study 33 SALyc-SDF2I pSASDF2I and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-SDF3I pSASDF3I and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-SI pSASI and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-DF1 pSADF1 and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-DF2 pSADF2 and pAC-LYC coexpressed in E. coli BL21(DE3) This study SALyc-DF3 pSADF3 and pAC-LYC coexpressed in E. coli BL21(DE3) This study Plasmids Description Source pSASDFI Ampr; trc promoter; genes dxs, ispD, ispF and idi; pBR322 ori This study pSASDFEI Ampr; trc promoter; genes dxs, ispD, ispF, idi and ispE; pBR322 ori This study pSASDF1I Ampr; trc promoter; genes dxs, ispDF1 and idi; pBR322 ori This study pSASDF2I Ampr; trc promoter; genes dxs, ispDF2 and idi; pBR322 ori This study pSASDF3I Ampr; trc promoter; genes dxs, ispDF3 and idi; pBR322 ori This study pSASDF1EI Ampr; trc promoter; genes dxs, ispDF1, idi and ispE; pBR322 ori This study pSADF1 Ampr; trc promoter; ispDF1; pBR322 ori This study pSADF2 Ampr; trc promoter; ispDF2; pBR322 ori This study pSADF3 Ampr; trc promoter; ispDF3; pBR322 ori This study pSAIspS Camr; araBAD promoter; ispS; p15A ori This study pSAHisDF1 Camr; T7 promoter; (His)6 tagged ispDF1; p15A ori This study pSAHisDF2 Camr; T7 promoter; (His)6 tagged ispDF2; p15A ori This study pSAHisDF3 Camr; T7 promoter; (His)6 tagged ispDF3; p15A ori This study pSASI Ampr; trc promoter; genes dxs and idi; pBR322 ori This study pAC-LYC Camr; crtE, crtI, and crtB under endogenous promoter; p15A ori Addgene plasmid 53270105 34 Genes Description Source dxs 1-deoxy-D-xylulose-5-phosphate synthase NCBI Gene ID: 945060 ispD 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase NCBI Gene ID: 948269 ispE 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase NCBI Gene ID: 945774 ispF 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase NCBI Gene ID: 945057 idi isopentenyl-diphosphate Delta-isomerase NCBI Gene ID: 949020 ispDF1 Codon optimized bifunctional 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase/2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (NR0032_N05) This study, USPTO PCT/CA2018/051073 ispDF2 Codon optimized bifunctional 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase/2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (NR0032_O07) This study, USPTO PCT/CA2018/051073 ispDF3 Codon optimized bifunctional 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase/2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (NR0037_N05) This study, USPTO PCT/CA2018/051073 ispS Isoprene synthase (Populus alba sp.) UniProtKB: Q50L36.1106 35 Non-natural protein fusions We constructed fusions with different linkers. The linkers used and their sequences are listed in Table 2.2. The linkers were added by PCR and generated by Gibson assembly. Table 2.2 Types of linkers used in the study and their sequences Linker type Polypeptide sequence (N terminus  C terminus) Reference Flexible Linker (FL) SLGGGGSAAA 107,108 Rigid Linker (RL) AEAAAKEAAAKEAAAKEAAAKEAAAKAAA 107,108 cjIspDF Linker (CJ) LPTPSFE 83 IspDF1 Linker (XL) RQRLRSAVAA This study CJ and XL linkers sequences were generated by aligning sequences of respective fusion enzyme with E. coli IspD and IspF. Homology models were built for the natural as well as non-natural chimeric fusions using SWISS-MODEL server. The non-natural fusions are listed in Table 2.3. The IspD and IspF domains of IspDF1 were also expressed separately. This was achieved by adding a stop codon (TAA) at the end of genetic sequence for domain ispD, taking out the genetic sequence for the linker, adding a RBS and a start codon (ATG) in frame with the genetic sequence for IspF. This enabled transcriptional level separation for the two domains. The genetic sequence coding for IspD domain is denoted as ispD1 with corresponding protein as IspD1. The genetic sequence coding for IspF domain is denoted as ispF1 with corresponding protein as IspF1. 36 Table 2.3 List of non-natural protein fusions Construction of plasmids and strains for analysis of non-natural fusions The non-natural fusions were cloned with other genes involved in the MEP pathway to assess their influence on the pathway flux. These constructs and strains are mentioned in Table 2.4. Table 2.4 Strains and plasmid expressing non-natural fusion proteins Strains Description SALyc-SDFLFI pSASDFLFI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDRLFI pSASDRLFI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDCJFI pSASDCJFI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDXLFI pSASDXLFI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDFLFEI pSASDFLFEI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDRLFEI pSASDRLFEI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDCJFEI pSASDCJFEI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDXLFEI pSASDXLFEI and pAC-LYC coexpressed in E. coli BL21(DE3) Fusion Enzyme Linker N-terminal protein/domain C-terminal protein/domain IspDFLF Flexible Linker (FL) E. coli IspD E. coli IspF IspDRLF Rigid Linker (RL) E. coli IspD E. coli IspF IspDCJF cjIspDF Linker (CJ) E. coli IspD E. coli IspF IspDXLF IspDF1 Linker (XL) E. coli IspD E. coli IspF IspDFLF1 Flexible Linker (FL) IspD domain of IspDF1 IspF domain of IspDF1 IspDRLF1 Rigid Linker (RL) IspD domain of IspDF1 IspF domain of IspDF1 IspDCJF1 cjIspDF Linker (CJ) IspD domain of IspDF1 IspF domain of IspDF1 IspDFLE Flexible Linker (FL) E. coli IspD E. coli IspE IspEFLF Flexible Linker (FL) E. coli IspE E. coli IspF 37 SALyc-SDFLF1I pSASDFLF1I and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDRLF1I pSASDRLF1I and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDCJF1I pSASDCJF1I and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SD1F1I pSASD1-F1I and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDFLF1EI pSASDFLF1EI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDRLF1EI pSASDRLF1EI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDCJF1EI pSASDCJF1EI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SD1F1EI pSASD1-F1EI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SDFLEFI pSASDFLEFI and pAC-LYC coexpressed in E. coli BL21(DE3) SALyc-SEFLFDI pSASEFLFDI and pAC-LYC coexpressed in E. coli BL21(DE3) Plasmids Description pSASDFLFI Ampr; trc promoter; genes dxs, ispDFLF and idi; pBR322 ori pSASDRLFI Ampr; trc promoter; genes dxs, ispDRLF and idi; pBR322 ori pSASDCJFI Ampr; trc promoter; genes dxs, ispDCJF and idi; pBR322 ori pSASDXLFI Ampr; trc promoter; genes dxs, ispDXLF and idi; pBR322 ori pSASDFLFEI Ampr; trc promoter; genes dxs, ispDFLF, idi and ispE; pBR322 ori pSASDRLFEI Ampr; trc promoter; genes dxs, ispDRLF, idi and ispE; pBR322 ori pSASDCJFEI Ampr; trc promoter; genes dxs, ispDCJF, idi and ispE; pBR322 ori pSASDXLFEI Ampr; trc promoter; genes dxs, ispDXLF, idi and ispE; pBR322 ori pSASDFLF1I Ampr; trc promoter; genes dxs, ispDFLF1 and idi; pBR322 ori pSASDRLF1I Ampr; trc promoter; genes dxs, ispDRLF1 and idi; pBR322 ori pSASDCJF1I Ampr; trc promoter; genes dxs, ispDCJF1 and idi; pBR322 ori pSASD1-F1I Ampr; trc promoter; genes dxs, ispD1, ispF1 and idi; pBR322 ori pSASDFLF1EI Ampr; trc promoter; genes dxs, ispDFLF1, idi and ispE; pBR322 ori pSASDRLF1EI Ampr; trc promoter; genes dxs, ispDRLF1, idi and ispE; pBR322 ori pSASDCJF1EI Ampr; trc promoter; genes dxs, ispDCJF1, idi and ispE; pBR322 ori pSASD1-F1EI Ampr; trc promoter; genes dxs, ispD1, ispF1, idi and ispE; pBR322 ori pSASDFLEFI Ampr; trc promoter; genes dxs, ispDFLE, idi and ispF; pBR322 ori pSASEFLFDI Ampr; trc promoter; genes dxs, ispEFLF, idi and ispD; pBR322 ori 38 Culture conditions Both isoprene and lycopene starter cultures were cultivated overnight at 30 °C in LB media (Sigma-Aldrich) containing appropriate antibiotic/s. Isoprene starter cultures were then diluted to 15 mL with the medium to OD600 of 0.2, induced with arabinose and/or IPTG; and allowed to grow for 24 h at 30 °C in 25 mL sealed glass tube. Lycopene starter cultures were diluted to 5 mL with the medium to OD600 of 0.2, induced with IPTG, and allowed to grow for 24 h at 30 °C in culture tubes in the dark. Isoprene analysis Isoprene analysis was performed on PerkinElmer Clarus 680 gas chromatograph and Perking Elmer Clarus SQ 8 T mass spectrometer (GC-MS). Since isoprene is volatile monoterpene, the sealed cultures were heated at 70 °C for 1 min and vortexed for 5 sec before sampling 200 µL of headspace using a gas-tight syringe. The standard curve for isoprene was prepared in a similar manner for quantification. HP-5MS capillary column (25 m long, 0.2 mm internal diameter, 0.33 µm film thickness; Agilent Technologies) was used, with helium (1 mL/min) as a carrier gas. The oven temperature program was 35 °C for 3 min, 25 °C/min to 200 °C and hold for 1min. The injector was maintained at 60 °C and 20:1 split ratio was maintained. Mass spectrum acquisition was carried out in SIR mode for m/z 68 and m/z 67 ions. Lycopene analysis Lycopene is an intracellular product. 2 mL of cell culture was centrifuged at 8000 rpm for 5 min and lycopene was extracted by extraction from the pellet with 1 mL acetone. Extraction was performed at 55 °C with intermittent vortexing for 20 min in reduced light condition. The acetone 39 suspension was centrifuged and filtered before analysis. Samples were analyzed on the PerkinElmer Flexar system equipped with Zorbax C-18 column (4.6 × 250 mm, Agilent Technologies) maintained at 30 °C. Samples were run with mobile phase consisting of 66% (v/v) methanol, 30% (v/v) tetrahydrofuran and 4% (v/v) water at 1 mL/min flow rate. Lycopene detection was done by monitoring absorbance at 474 nm wavelength using a photodiode detector. 2.3 Results Discovery of three IspDF fusions Soil metagenome sequences were screened and led to the discovery of novel bifucyional fusions of IspD and IspF. They were isolated from fosmids NR0032_N05, NR0032_O07 and NR0037_N05 and the corresponding genes were annotated as ispDF1, ispDF2 and ispDF3 respectively. The translated polypeptides were annotated as IspDF1 (41.6 kDa), IspDF2 (42.1 kDa) and IspDF3 (40.2 kDa) respectively. The sequences of the novel enzymes are listed in Appendix A These genes were tagged for affinity-based separation and expressed in E. coli BL21(DE3) using 0.5 mM IPTG as an inducer. Figure 2-1 is the image of the SDS/PAGE gel stained with Coomassie dye. Desired bands were seen on the gel but the expression levels of IspDFs were low. Insoluble cell debris were denatured and analyzed, and it was realized that all three fusions formed inclusion bodies. Sequences of IspDF1, IspDF2 and IspDF3 were aligned with E. coli IspD, IspF and cjIspDF (Table 2.5 and Figure 2-2). The discovered enzymes were more similar to the native monofunctional enzymes in E. coli. When aligned against cjIspDF83, more differences were observed. Though most of the residue functions were conserved among all five (Figure 2-2), the dissimilarity existed in clusters. The amino acid region between 220 and 250 residues was highly 40 variable and was involved in linking both the domains. Other dissimilar clusters were observed in IspD domain of the fusion. All three IspDFs discovered have novel sequence and are not reported. Figure 2-1 Expression of the novel IspDFs as (His)6 tagged protein expressed in E. coli BL21(DE3) and their analyses on SDS/PAGE. Lanes 1 and 5: total and purified IspDF1 extract respectively, lanes 2 and 6: total and purified IspDF2 extract respectively, lanes 4 and 7: total and purified IspDF3 extract respectively, lanes 2 and 8: protein ladder. The bands corresponding with the specific protein are highlighted with red box. Table 2.5 Protein alignment analysis of the bifunctional enzymes against E. coli IspD-IspF and cjIspDF using the online BLASTN search tool Bifunctional enzymes % Query cover when aligned with E. coli IspD, IspF % Sequence similarity when aligned with E. coli IspD, IspF % Query cover when aligned with cjIspDF % Sequence similarity when aligned with cjIspDF IspDF1 97 40.81 94 29.71 IspDF2 99 40.72 97 29.75 IspDF3 98 41.60 99 32.20 41 Figure 2-2 Protein sequence alignment generated from the Centre for Genomic Regulation (CRG) M-Coffee tool. IspD-IspF is the sequences for E. coli IspD and IspF. 42 Each domain of the fusion enzymes was aligned against E. coli IspD and E. coli IspF (Table 2.6). The IspF domains of the fusions share greater sequence similarity with E. coli IspF than the similarity between IspD domain and E. coli IspD. This observation is consistent with the similarity reported for cjIspDF with E. coli native enzymes66. IspF domain of cjIspDF shares 48 % sequence similarity with E. coli IspD whereas IspD domain shares 25 % similarity with E. coli IspD. Table 2.6 Protein alignment analysis of each domain of the bifunctional enzymes against corresponding E. coli monofunctional enzymes using the online BLASTN search tool Bifunctional enzymes % Query cover when IspD domain aligned with E. coli IspD % Sequence similarity when IspD domain aligned with E. coli IspD % Query cover when IspF domain aligned with E. coli IspF % Sequence similarity when IspF domain aligned with E. coli IspF IspDF1 94 35.71 90 56.38 IspDF2 100 37.55 89 49.35 IspDF3 98 35.93 96 51.30 When the domains of fusions were aligned against cjIspDF domains, a similar trend was observed (Table 2.7). 43 Table 2.7 Protein alignment analysis of each domain of the bifunctional enzymes against corresponding cjIspDF enzyme domains using the online BLASTN search tool Bifunctional enzymes % Query cover when IspD domain aligned with IspD domain of cjIspDF % Sequence similarity when IspD domain aligned with IspD domain of cjIspDF % Query cover when IspF domain aligned with IspF domain of cjIspDF % Sequence similarity when IspF domain aligned with IspF domain of cjIspDF IspDF1 95 23.77 86 42.36 IspDF2 97 24.89 86 41.72 IspDF3 95 26.89 95 43.14 44 Construction of MEP pathway operon Enzymatic steps catalyzed by Dxs, IspD, IspF and Idi are the rate-controlling steps of the MEP pathway28 in E. coli. The same operon was reconstructed (pSASDFI) and analyzed for protein expression. The soluble protein samples were run SDS/PAGE gel and stained with Coomassie dye. Figure 2-3 SDS/PAGE image of the soluble protein fraction of pSASDFI. Lane 1: E. coli BL21(DE3), lane 2: protein ladder, lane 3 and 4: SASDFI. The bands corresponding to protein are: Dxs (band a, 68.2 kDa), IspD (band b, 25.7 kDa), IspF (band d, 16.9 kDa) and Idi (band c, 21.2 kDa). SASDFI was tested for activity towards isoprene and lycopene production by co-expressing with downstream pathway (pSAIspS and pAC-LYC respectively). The clone expressing Dxs and Idi (pSASI) was constructed to account for the influence of IspD and IspF on MEP pathway flux improvement. 45 Figure 2-4 Influence of rate-limiting steps on MEP pathway flux. (a) Lycopene production, (b) Isoprene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. SALyc and SAIso made the corresponding terpenoid at very low yield (Figure 2-4). These strains reflect the native expression level of the MEP pathway. Induction did not have a substantial influence on terpenoid production. IPTG induction for SAIso had a negative impact on cell growth and hence shows higher normalized yield. Higher IPTG induction levels were detrimental to lycopene production and had a negative influence on growth. Overexpression of Dxs and Idi (strains SALyc-SI and SAIso-SI) produced 22-fold and 12-fold more terpenoid respectively. Additional expression of IspD and IspF (strains SALyc-SDFI and SAIso-SDFI) further enhanced the terpenoid production by 47-fold and 15-fold respectively. Uninduced cultures of SALyc-SI and SALyc-SDFI still produced lycopene at a higher yield than that of SALyc. 0 5 1003060901200100200300400Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer0 50.00.51.01.52.002468Isoprene Produced (mg/L/OD)Isoprene Produced (mg/L)0 µM 50 µM Normalized Titer(a) (b)46 Influence of novel fusion enzymes on the MEP pathway All three fusions exhibited different effects on isoprene and lycopene production (Figure 2-5). SALyc-SDF1I and SAIso-SDF1I were the best performers. There was 20% and 75 % improvement in lycopene and isoprene production respectively for IspDF1 strains. The IspDF2 and IspDF3 versions lowered the titer. OD600 for strains were in a similar range. IspDF1 variants showed higher normalized titer which means the catalytic throughput was improved as well. SALyc-SDF1I was tested at IPTG induction concentrations of 75 µM and 100 µM, but the titer declined, and the maximum titer was obtained at 50 µM IPTG concentration. To assess the influence sole contribution from IspDFs, strains SAIso-DF1, SAIso-DF2 and SAIso-DF3 were tested for isoprene productions; and strains SALyc-DF1, SALyc-DF2 and SALyc-DF3 were tested for lycopene production. All these six strains made respective terpenoid in the levels equal to SAIso and SALyc (data not shown). The induction had no effect on the terpenoid titer. 47 Figure 2-5 Influence of novel IspDF fusions on MEP pathway flux. (a) Lycopene production, (b) Isoprene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Homology models for the fusions were generated by SWISS-MODEL using cjIspDF as a template (Figure 2-6). All four fusions have conserved subunit structures. IspDF1 and IspDF3 align well with cjIspDF but IspDF2 has a longer linker. The active sites of the subunits are located at opposite ends. The putative linker sequences are: EAIARGTGERAVGERAA for IspDF2 and ERLIGARNTAGAM for IspDF3. Since, IspDF1 improved the terpenoid titer, it was used for further study. 0 2 4 6 8 10 120.00.81.62.4024681012Isoprene Produced (mg/L/OD)Isoprene Produced (mg/L)0 µM 50 µM Normalized Titer0 2 4 6 8 10 12 14 16040801200100200300400500Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer(a) (b)48 Figure 2-6 Homology models for the fusion proteins generated by SWISS-MODEL tool. (a) cjIspDF66, (b) IspDF1, (c) IspDF2 and (d) IspDF3. The IspD domain is in pink, the IspF domain is in blue and linker is in green. The N-terminal residue is colored black and C-terminal residue is colored orange. Role of IspE IspE is reported to influence the flux by associating with IspD and IspF66. The association complex then assists the efficient transfer and conversion of metabolites from MEP to MEcPP. I investigated this phenomenon for lycopene production by testing the recombinant E. coli strain expressing five enzymes Dxs, IspD, IspF (or IspDF), IspE and Idi. For both SALyc-SDFEI and 49 SALyc-SDF1EI had lower lycopene titers than SALyc-SDFI and SALyc-SDF1I respectively (Figure 2-7). The percent loss in flux on IspE overexpression was more evident for IspDF1 clone than monofunctional native enzyme clone. This effect was a summation of the lower rate of lycopene as well as lower cell growth rate. The OD600 in IspE clones was remarkably lower (by 20-60 %). SALyc-SDFEI cultures had higher variable growth reflecting in wider error bars. Figure 2-7 Effect of IspE overexpression on lycopene production. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Effect of linkers on IspDF1 activity To evaluate the role of the linker in the enhancement of flux in SALyc-SDF1I, I replaced the putative linker sequences with three types of linkers. First is the linker identified from cjIspDF. Second is ‘FL’ that is glycine and serine linker and imparts flexibility to the domains. The third is 0 2 4 6 8 10 12 14 16040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer50 ‘RL’ that forms an α-helix and restricts the free movement and giving rigidity to the conformation. The effect of the linker was tested in strains with and without IspE overexpression. The non-natural linkers did not improve the overall titers of lycopene (Figure 2-8) but influenced cell viability and lowered OD600 for the cultures. Normalized titers were highest for SALyc-SDRLF1I followed by SALyc-SDCJF1I. The clone with flexible linker displayed lowest lycopene titers in both the sets. Figure 2-8 Linkers for IspDF1 and their effect on MEP pathway flux. (a) Strains overexpressing Dxs, IspDF chimeras and Idi, (b) strains overexpressing Dxs, IspDF chimeras, IspE and Idi. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. 0 2 4 6 8 10 12 14 1603060900100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer0 2 4 6 8 10 12 14 16040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer(a) (b)51 Effect of linkers on non-natural fusions of monofunctional IspD and IspF Likers in section 2.3.5 above failed to improve the lycopene titers but had a positive impact on the normalized titers. This means that the linkers improved the flux at the cost of cell growth. The same linkers along with the natural linker of IspDF1 were then employed to link E. coli IspD and IspF. Any type of linkers did not perform better than the monofunctional enzyme construct for lycopene production (Figure 2-9). For strains in Figure 2-9(b), the lower normalized titers were the result of higher OD600. This suggests overall carbon flux channeling towards cell growth metabolisms. Whereas, for strains depicted in Figure 2-9(a), the fusions had a negative impact on lycopene products without the substantial effect of cell growth. Figure 2-9 Linkers for non-natural fusions of E. coli IspD and IspF; and their effect on MEP pathway flux. (a) Strains overexpressing Dxs, IspDF chimeras and Idi, (b) strains overexpressing Dxs, IspDF chimeras, IspE and Idi. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. 0 2 4 6 8 10 12 14 16040801200100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer0 2 4 6 8 10 12 14 16040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer(a) (b)52 Effect of XL on non-natural fusions of monofunctional E. coli IspD and IspF Since the strains exhibited a mixed response to CJ, FL and RL linkers, fusions of IspD and IspF with the putative linker of IspDF1 were constructed. These fusions lowered the MEP flux and further decreased the lycopene production (Figure 2-10). This effect was pronounced for SALyc-SDXLFI than SALyc-SDXLFEI. Figure 2-10 Linkers for non-natural fusions of E. coli IspD and IspF on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Expression of domains of IspDF1 as IspD1 and IspF1 The XL linker’s negative impact on the pathway flux suggested the need to study the domains of IspDF1 in isolation (Figure 2-11). The separation of the domains as individual enzymes had a more pronounced effect on SALyc-SD1F1EI. 0 2 4 6 8 10 12 14 16040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer53 Figure 2-11 Effect of domain separation of IspDF1 on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Non-natural fusions of IspE and their effects on MEP pathway flux To evaluate the cause behind the natural existence of fusions of enzymes that catalyze non-consecutive steps in the MEP pathway, I constructed non-natural fusions of IspE. The fusions were constructed using the flexible linker. The linking strategy was kept similar to that of natural IspDFs. The IspDE fusion was constructed by linking C-terminus of IspD to N-terminus of IspE. And, the IspEF fusion was constructed by linking C-terminus of IspE to N-terminus of IspF. Figure 2-12 shows that IspDE fusion exhibited a 20 % improvement in lycopene production compared to SALyc-SDFI and 2.3-fold improvement than SALyc-SDFEI. Whereas, IspEF fusion lowered the lycopene production substantially. 0 2 4 6 8 10 12 14 16040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer54 Figure 2-12 Non-natural fusions of IspE and their effect on MEP pathway flux. The IPTG concentrations used for induction are denoted in the legends. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Categorical inferences Figure 2-13 summarizes the results obtained so far. It is a comparison plot for different constructs with the highest titer and normalized titer values. The blank places denoted by ‘-‘. 0 5 10 15 20040801201600100200300400500600Lycopene Produced (mg/L/OD)Lycopene Produced (mg/L)0 µM 25 µM 50 µM Normalized Titer55 Figure 2-13 Categorical comparison of lycopene production for the various linker. *IspD and IspF domains of IspDF1 are separated to be translated as two polypeptides. 2.4 Discussions Influence of native Dxs, IspD, IspF and Idi on MEP pathway flux The downstream lycopene production genes (crtE, crtB, crtI) are under the control of an endogenous promoter and MEP pathway genes are under the control of trc promoter that is reported to be leaky109–111. Due to these reasons, lycopene cultures at no induction produced higher 56 lycopene than that of the base strain SALyc. Higher normalized titers in both lycopene and isoprene fermentation indicate an abundance of C5 precursor metabolites- IPP and DMAPP that are shuttled to respective downstream terpene synthesis pathway. To study fusions and role of linkers, it was necessary to construct the basal operon overexpressing Dxs, IspD, IspF and Idi that was reported to increase the taxol yield28. This strain containing plasmid pSASDFI served as the basis for comparison in this study. Some reports emphasized overexpression of Dxs and Idi only for improvement of MEP pathway flux112,113 and results of my study (Figure 2-4) showed that additional overexpression of IspD and IspF improved the titers for lycopene by 80% and that of isoprene but 35 %. The micro-aerobic environment during isoprene cultures could be responsible for the disparity in titers as it is the highly oxygen-limited environment. The Lycopene titers obtained in SALyc-SDFI are comparable to the titers reported in literature114,115. Overall, the pSADFI operon improved lycopene production by 47-fold and isoprene titers by 15-fold compared to pSALyc and pSAIso strains; and the strategy proved to be effective in eliminating bottlenecks in the MEP pathway. IspDF fusion activity and the influence of IspE on MEP pathway flux Dxs is a gatekeeper gene in the MEP pathway and Idi catalyzes the terminal step maintaining equilibrium in IPP and DMAPP concentrations required for the downstream pathway of terpenoid biosynthesis. Hence, the operon overexpressing only IspD and IspF as well as IspDFs did not influence the terpenoid titers. Production of terpenoids by SAIso-DF1, SAIso-DF2, SAIso-DF3, SALyc-DF1, SALyc-DF2 and SALyc-DF3 were not significantly different than the strains with no MEP pathway overexpression (data not shown). Hence it was necessary to include genes dxs and idi in further experiments to study the influence of intermediary steps. 57 Improvement in the flux through the pathway due to IspDF1 expression in pSASDF1I operon can be attributed to the role of the linker imparting physical features (like flexibility or catalytic site proximity/substrate channeling) to the catalytic domains; and/or, higher stability and/or activity of IspDF1 than the native monofunctional enzymes. The IspF domain of IspDF1 has the highest similarity to the E. coli IspF than that of IspDF2 and IspDF3. The intensity of influence of IspDF1 overexpression in lycopene strain was different than the isoprene strain. Since, IspE catalyzes the step between IspD and IspF, further investigation was carried out to evaluate the role of IspE in the catalytic cascade. IspE catalyzed step is not reported to be the bottleneck in the pathway and its overexpression exerted metabolic stress and lowered the lycopene titers. The stress effect was dominant in SALyc-SDF1EI even though it expressed only four recombinant proteins versus five recombinant proteins in SALyc-SDFEI. This result highlighted the existence of factor/s other metabolic stress. Study on linker types The first factor studied was the role of the linker. The type of fusion and linker affected only their activity and did not contribute to variability in protein expression levels. This was verified by enzyme expression and analysis on SDS/PAGE (data not shown). Flexible linker was chosen to impart mobility to the domains and rigid linker was chosen that forms a long helix restricting movements of the domains. Linker from cjIspDF was employed as well. For the non-natural IspDF1 fusion, the C flux was diverted more to the MEP pathway and away from growth resulting in higher normalized lycopene titers but lower total lycopene production. SALyc-SDRLF1I was best performing strain with 22% higher normalized titers than SALyc-SDF1I and 33% higher normalized titers than the basal strain SALyc-SDFI. This suggested that the rigidity in 58 the conformation of the fusion had a positive impact on the catalytic activity. Homology modeling IspDRLF1 was inconclusive since the templates could not accurately replicate the folding of the linker. On the other hand, when IspE was overexpressed (strain SALyc-SDRLF1EI), the production decreased by 30 % and the normalized titers lowered by 80 %. But the OD600 of SALyc-SDRLF1EI was 50 % higher than SALyc-SDRLF1I. Since the SALyc-SDRLF1EI expresses 4 heterologous enzymes, the effective quantities of the individual enzyme are lower than that in SALyc-SDRLF1I that expresses 3 heterologous enzymes. Hence the MEP pathway flux was lower, and the overall C flux was diverted to biomass generation. To deduce this effect further, construction and comparison were made with non-natural fusions of E. coli IspD and IspF. In these cases (Figure 2-9), co-localization of the activities had a negative impact on lycopene production as well as normalized titers. But, in these cases the overall OD600 of strains overexpressing IspE was 10-50% lower than their corresponding variants not overexpressing IspE. This prompted the involvement of IspE beyond its influence on the health and growth of the cell. Moreover, the putative linker of IspDF1 when used to link E. coli IspD and IspF, exhibited similar effects as other non-natural fusions. Influence of IspDF1 domain separation on MEP pathway flux The chimeric enzyme with RL type linker so far exhibited the maximum flux through the MEP pathway at an expense of cell growth. This prompted to re-evaluate necessity for linker and domain co-localization. The prevailing theory of organization of fusion of enzymes improves the rate of reaction cascade is by lowering the substrate diffusional limitations and substrate channeling. But the recent evidence shows that the dynamics of fusions on a metabolic cascade is more complex than previously assumed116. It is not simply the proximity between enzymes that 59 enhances the initial reaction rate; rather, colocalization increases the local concentration of enzymes. This therefore increases the chance that a diffusing substrate will interact with an active site cavity117. IspD1 and IspF1 retained the individual activities. Strain SALyc-SD1F1I had 25 % lower lycopene production but the OD600 was lower by the same factor as well. Hence the overall flux and the normalized titers were similar. SALyc-SD1F1EI had 30 % lower OD600 and displayed an 82 % increase in lycopene production. Both these observations factored to three-fold improvement in normalized lycopene titers for SALyc-SD1F1EI than SALyc-SDF1EI. Though the overall lycopene titers remained lower than SALyc-SDF1I due to lower availability of copies of enzyme because of longer operon; improvement in the normalized titers bolstered the observation of higher stability and activity of IspDF1. Improvement in the flux on IspDE fusion The absence of any literature on the fusion of IspE is noticeable in contrast to many discoveries of IspDFs. The role of fusion of enzymes catalyzing non-consecutive steps in the pathway and role of intermediary step enzyme is not only highly debated but also rather unforeseeable. I tried to unravel it by constructing non-natural fusions of IspE. The performance of IspDE fusion was many folds better than IspEF fusion. In fact, IspDE fusion exhibited 2.3-fold improvement in lycopene production and 20 % improvement in the normalized titers than that of SALyc-SDFEI. The OD600 for SALyc-SDFLEFI was doubled as well. Whereas, IspEF fusion decreased the lycopene production at least 65 % normalized titers by 85 % than of SALyc-SDFEI. The strain SALyc-SDFLEFI was the best performing strain for lycopene production and second 60 best in MEP pathway flux after SALyc-SDRLF1I. This was due to the fact that the individual domains of IspDF1 are higher active than E. coli native IspD and IspF. In conclusion, I was able to successfully engineer higher active ortholog of MEP pathway enzyme by fusion IspDF1. I decoded it by the strategy to study linkers where non-natural IspDE fusion performed as well as the IspDF1. This posed the question on the plausible role of IspE in the enzyme cascade. The positive effect of active site co-localization should have worked fairly equally for IspDE and IspEF since, they are the fusions of consecutive steps in the pathway. Rather, IspEF underperformed 7-fold than IspDE for lycopene production as well as 8.5-fold for normalized titers that are indirect measures of MEP pathway flux. These results pose questions about the canonically reported MEP pathway for a cascade of IspD, IspE and IspF catalyzed steps. 61 Chapter 3: Study of MEP pathway biochemistry 3.1 Introduction The work presented in chapter 2 of this thesis put forth the question on the benefit of the natural existence of fusion protein (IspDF) that catalyzes non-consecutive steps in the MEP pathway. Clearly, the reason isn’t substrate channeling. If it was so, IspDE or IspEF fusions have been common too. Moreover, the non-natural fusion IspDE was higher active than IspEF fusion. If the reason underlies in ease of complexation to form multicatalytic complex66 of IspD, ispE and IspF, published work in this area has never reported IspE catalyzed reaction as the rate limiting66,85. This brought our attention to reconsider the MEP pathway steps. The cascade of IspD, IspE and IspF catalyzed steps attracted our attention. These discoveries were the result of radiolabelling individual substrates and testing the reactions in isolation in vitro. Out of the three, IspF is reported to be promiscuous catalyzing cyclization of CDP-ME to 2-C-Methyl-D-erythritol-3,4-cyclophosphate43. Though this product was regarded as in vitro artifact without biosynthetic relevance to the MEP pathway, the enzyme’s promiscuous nature cannot be ignored. Kinetics of MEP pathway cascade from MEP to MEcPP The recent study published gives some insights about the MEP pathway flux118 summarized in Table 3.1. These estimations were based on experiments carried out using glucose as a substrate and E. coli cell lysate. Though they are not the in vivo measurements, they give an overview of the three-enzyme cascade. Fluxes through IspD and IspF are lower many folds compared to that of IspE though Kcat value for IspE is lower than IspD, the higher availability of IspE drives higher flux. 62 Table 3.1 Enzyme amount and kinetics for MEP pathway steps (data extracted from118) Enzyme Kcat (min-1) Amount per cell (molecules) Maximum flux per cell (molecules min-1) IspD 2940 50 ± 8 1.5 × 105 IspE 1010 208 ± 22 2.1 × 105 IspF 60 111 ± 13 6.7 × 103 Moreover, each enzyme association complex reported66 contains 6 molecules each of IspD, IspE and IspF. But the amounts reported per cell are not equal. In fact, the rough ratio of molecules estimated (Table 3.1) is 1:4:2 for IspD:IspE:IspF distorting the stoichiometry required complexation. The natural fusion enzyme IspDF1 exhibited higher flux as well as terpenoid yield due to the higher activity of each domain that was clear from non-natural fusion IspDRLF1 and from the separation of the domains to IspD1 and IspF1. Another of my study revealed that IspDFLE fusion improved the pathway flux but not IspEFLF. The interpretations from the literature as well as observations of my past experiments directed me to study the pathway steps in detail with respect to the order of occurrence reported. Canonical MEP pathway and plausible bifurcation step Conversion of MEP to MEcPP happens through three steps in the order of citidylyl transfer, phosphorylation and intramolecular cyclization. In the canonical MEP pathway reported (Table 3.1(a)), MEP enters IspD catalytic site and gets converted to CDP-ME. CDP-ME diffuses out of the IspD in the bulk and enters IspE catalytic site to get converted to CDP-MEP. CDP MEP then 63 diffuses back in the bulk and enters IspE active site to be converted to MEcPP. These steps involve many diffusional steps. Figure 3-1 Schematic for the flow of metabolites through the MEP pathway for IspDF, IspE. (a) Canonical MEP pathway, (b)MEP pathway bifurcation. A: MEP, B: CDP-ME, C:CDP-MEP, D:MEcPP, B’: ME-2,4-PP We postulate that IspE can act on MEP to phosphorylate 2-OH group to form 2-Methyl-erythritol-2,4-diphosphate (ME-2,4-PP) as an alternate intermediate. ME-2,4-PP will then be acted upon by IspD to for CDP-MEP and resume following steps reported in the canonical pathway (Figure 3-1(b) and Figure 3-2). This route involves fewer diffusional steps in case of fusions and are also aligned with my results of section 2.3.9 (page number 53) of this thesis. 64 Figure 3-2 Bifurcation in the MEP pathway with the promiscuity of IspE. The pathway highlighted with blue arrow is the canonical MEP pathway and the step highlighted by yellow arrow marks the bifurcation step from MEP. In this work, I systematically evaluated the feasibility of conversion of MEP to ME-2,4-PP by IspE. 3.2 Materials and methods Strains, plasmid and genes All strains, plasmids and genes used in this study are listed (Table 3.2). Genes ispE was amplified from E. coli strain K12 genome. All constructs were sequenced and verified from Genewiz Inc., USA. IspE protein was tagged with 6×His residues for affinity-based purification. MEPCDP-MECDP-MEP MEcPPME-2,4-PPPPiCTPIspDATPADPIspECMPIspFIspE IspDPPiCTPADP/AMPATP/ADP65 Table 3.2 Strains, plasmids and genes Strains Description Source E. coli DH5α Cloning strain New England Biolabs (# C2987) E. coli BL21(DE3) Expression strain New England Biolabs (#C2527) E. coli strain K12 Gene amplification Sigma-Aldrich (#EC1) SAHisIspE E. coli BL21(DE3) transformed with pSAHisIspE This study Plasmid Description Source pSAHisIspE Camr; T7 promoter; (His)6 tagged ispE; p15A ori This study Gene Description Source ispE 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase NCBI Gene ID: 945774 Media and growth conditions E. coli DH5α was used for plasmid construction and cultivated at 37 °C in Luria Bertani (LB) media (purchased from Sigma-Aldrich). E. coli BL21(DE3) was used as an expression host. The successful transformant of SAHisIspE was selected on LB agar plate containing 50 µg/mL chloramphenicol. For expression of IspE, a single colony from the plate was inoculated in LB media supplemented with chloramphenicol and grown at 37 °C for 6 h. This inoculum was suitably diluted with fresh media to the OD600 of 0.2 and grown at 37 °C. When the OD600 reached 0.8, the culture was induced with 0.5 mM IPTG and allowed to grow at 30 °C overnight. All the chemicals were purchased from Sigma-Aldrich. 66 Protein extraction and IspE purification The cells were harvested, and soluble proteins were extracted from the cell pellet. Lysis buffer consisted of 50 mM Tris-HCl (pH 8.0), 10 % (v/v) glycerol, 0.1 % (v/v) Triton X100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM magnesium chloride and 3 units/mL DNAase. The samples were sonicated to completely lyse cells. IspE was purified from the total soluble protein fraction by immobilized metal-affinity chromatography (IMAC) on Qiagen Ni-NTA Agarose (Qiagen Cat No./ID: 30210) using protocol provided by the supplier. IspE purification was performed using affinity-based technique utilizing Ni-NTA resin columns supplied by Qiagen. The purification was performed as per the supplier’s protocol and the purified protein solution was exchanged with 0.1 mM Tris-HCl and concentrated by ultrafiltration. Protein concentration was measured by the Bradford method using bovine serum albumin as a standard. The protein samples were stored at -20 °C until use. IspE in vitro reactions In vitro reaction consisted of 0.1 mM Tris HCl, 5 mM DTT, 10 mM MgCl2, ATP (or ADP), MEP, IspE enzyme. Chemicals and standards were procured from Thermo Scientific. The reaction was initiated by addition of substrate and allowed to proceed at 37°C 85,119. The reaction mixture was sampled, and the reaction was arrested by vortexing and addition of equal volume of methanol. The samples were then desalted using polyethersulfone (PES) membrane filters with 3 kDa MWCO (VWR International). 67 Bioluminescent assay for ATP consumption Initial reactions were performed using ATP as a phosphate donor. The course of the reaction was monitored using ATP Bioluminescent Assay Kit (Sigma-Aldrich) using the supplier’s protocol. HPLC analysis of ATP, ADP and AMP Further analysis of reaction samples to analyze products of ATP was performed on PerkinElmer Flexar HPLC consisting of Zorbax C-18 column (4.6 × 250 mm, Agilent Technologies) maintained at 25 °C. Samples were run with 1 mL/min mobile phase containing solvent A: 10 mM ammonium acetate and solvent B: acetonitrile. Gradient elution conditions were: 95 % A and 5 % B at 0 min; changed to 10 % A and 90 % B in 10 min; run for next 4 min; gradient back to 95 % A and 5 % B in 4 min. Detection was done at 254 nm. This analysis could neither detect MEP not its phosphorylated product. LC-MS analysis of MEP and ME-2,4-PP The reaction components were further analyzed on LC Agilent 1100 /MSD Trap XCT plus system (Agilent Technologies) in negative mode consisting of Zorbax SB-C18 column (4.6 × 150 mm) maintained at 25 °C. Samples were run with 1 mL/min mobile phase containing solvent A: 0.2 % (v/v) formic acid solvent B: acetonitrile. Gradient elution conditions were: 95 % A and 5 % B for 1 min; changed to 10 % A and 90 % B in 5 min; run for next 1 min; gradient back to 95 % A and 5 % B in 10 sec. The ESI temperature was set at 350 °C with nebulizer set at 60 psi. Scans were performed in negative mode. 68 Computational analysis The analysis was done against IspD (PDB ID 1I52), IspE (PDB ID 1OJ4) and IspF (PDB ID 1JY8). The PDB The structures of all molecules that participate in the catalytic cycle were prepared in Marvin120. The molecules were then docked into the active site of the enzymes using AutoDock Vina121. The input to the docking calculation comprised an initial set of atomic coordinates, and the program outputs putative poses of the molecule within the active site. A corresponding Gibbs free energy of binding (ΔG) was also computed for each pose. In order to identify the pose that yields the global minimum for ΔG, the most stable pose was selected, and its coordinates were re-used as the initial value for a subsequent docking calculation. This methodology was reiterated until an unchanging pose was outputted. 3.3 Results Protein expression and quantification The IspE protein was successfully expressed and run on SDS/PAGE. (His)6-IspE is 32.9 kDa protein that can be seen in the image of a gel stained with Coomassie dye (Figure 3-3). 69 Figure 3-3 IspE expression and analysis on SDS/PAGE. Lane 1: Total soluble protein fraction of E. coli BL21(DE3), lane 2: total soluble protein fraction of SAHisIspE induced with 0.5 mM IPTG, lane 3: protein ladder, lanes 4 and 5: purified IspE. The bands corresponding to IspE are highlighted with red box. Analysis of IspE reaction by ATP bioluminescent assay In vitro reactions of IspE were carried out using MEP and ATP/ADP as substrates. Four sets of conditions were studied (Table 3.3). Table 3.3 Reaction conditions for different experimental sets Set No. IspE concentration (µM) MEP concentration (µM) ATP concentration (µM) ADP concentration (µM) 1 6 500 150 0 2 30 500 150 0 3 30 200 1000 0 4 30 200 0 1000 70 The set 1 of the reactions was carried out keeping the molar ratio of the ATP (150 µM) to MEP (500 µM) at 1:3.3 and IspE concentration was kept at 6 µM. The reaction was monitored using the bioluminescent assay. The assay is two-step procedure that measures both ATP and ADP based on luciferase activity. The drop in relative luminescent units (RLU) corresponds to the utilization of ATP whereas, ADP/ATP ratio gives ADP amount present indirectly. The course of this reaction was monitored for 4 h. The RLU drop over time indicated consumption of ATP in the reaction (Figure 3-4) but the proportional increase in the ADP concentration was not observed. The ADP concentration suddenly increased after 2 and soon reached a plateau. Both ATP and ADP amounts reached saturation after 3 h. Figure 3-4 Bioluminescent assay monitoring ATP and ADP in the IspE reactions. 00.20.40.60.810204060801000 60 120 180 240 300ADP/ATP RatioDrop in RLU (%)Time (min)Drop in RLU (%)ADP/ATP ratio71 HPLC analysis of IspE reactions The bioluminescent analysis showed consumption of ATP, but non-proportional ADP buildup. To investigate this further, the set 1 experimental samples were analyzed by HPLC. It revealed a conversion of ATP to AMP and the ADP buildup only occurred at the later stages of reaction (Figure 3-5). These results were in agreement with the bioluminescence analyses. The reaction did not go to completion and 75 % of ATP was consumed at the end of 3 h. The reaction rate is very low and the canonical reaction of conversion of CDP-ME to CDP-MEP is reported faster85. The initial rate of reaction was calculated from the ATP consumption data. The rate for this reaction was observed to be 0.781 µM.min-1. The reaction samples without enzyme but with ATP, ADP and AMP were analyzed as well to assess temperature and pH-dependent degradation of the phosphates. No noticeable degradation was observed for 3 h at reaction conditions. Figure 3-5 HPLC analysis of set 1 reactions to study ATP consumption. 03060901201500 30 60 90 120 150 180Concentration (µM)Time (min)ATP ADP AMP72 Hence, the set 2 experiments were designed with exactly the same parameters but IspE concentration of 30 µM. 75 % ATP consumption was achieved in 1 hour and ATP was rapidly converted to AMP and ADP built over time (Figure 3-6). The rate of ATP conversion increased 2.3-fold to 1.795 µM.min-1. Figure 3-6 HPLC analysis of set 2 reactions with higher IspE concentration to study ATP consumption. The first two steps involved ATP as the limiting reactant, set 3 reactions were planned with ATP to MEP ratio of 5:1 so that ATP is no longer a limiting reactant. The ATP consumption reached a maximum in the first 90 min of the reaction (Figure 3-7) and the initial rate was 2.521 µM.min-1. But the total conversion decreased to 30 %. Conversion of ATP to AMP was faster than that to ADP. 03060901201500 30 60 90 120 150 180Concentration (µM)Time (min)ATP ADP AMP73 Figure 3-7 HPLC analysis of set 3 reactions when MEP is rate limiting reactant to study ATP consumption. This suggested sequential phosphorylation of ATP to first ADP and ADP to AMP. To assess the role of ADP as a phosphate donor for IspE reaction, a reaction set 4 were planned with only ADP and MEP as substrates in the ratio of 5:1. It was observed that ADP is utilized and converted to AMP (Figure 3-8). The rate of reaction was 2.967 µM.min-1 which is 17 % higher than that of utilization of ATP as phosphate donor. 020040060080010000 30 60 90 120 150 180Concentration (µM)Time (min)ATP ADP AMP74 Figure 3-8 HPLC analysis of set 4 reactions when ADP is used as a phosphate donor. The HPLC analyses gave insights about the dynamics of the reaction but failed to detect MEP and ME-2,4-PP. LC-MS analyses of IspE reaction mixture LC-MS analysis was performed on set 3 and 4 reaction samples. MEP peak was characterized by the standard. ME-2,4-PP chemical synthesis for the standard was attempted but failed several times. Hence, the online tool Competitive Fragmentation Modeling for Metabolite Identification (CFM-ID) was used to predict probable fragmentation pattern for the product. The following spectra (Figure 3-9) was seen that closely corresponded to peaks predicted for ME-2,4-PP (highlighted in the figure). The 296.64 is molecular ion (M-) peak and 280.71 and 264.71 are the major fragments. 020040060080010000 15 30 45 60Concentration (µM)Time (min)ADP AMP75 Figure 3-9 Mass spectra of the peak identified as ME-2,4-PP in IspE reactions. Additionally, ME-3,4-PP spectral predictions prompted fragments at m/z 153 and 155 corresponding to carbonyl (Figure 3-10(a)) and hydroxy (Figure 3-10(b)) monophosphates respectively. These tautomeric rearrangements are only possible when the phosphate is present at C3 position and; a methyl and a hydroxy group at the C2 position. The spectra of the sample lacked these fragments and hence confirm that ME-3,4-PP adduct is absent. Figure 3-10 The major mass fragments of ME-3,4-PP. 050001000015000200002500068.1196.97121.74144.87164.84180.8201.76216.8233.72261.88272.74281.7294.72315.71329.2343.11355.4366.92378.72387.71397.03410.61418.95426.5435.54447.68454.73464.54471.49480.43490.66498.67Ion countsm/z296.64264.71280.71CH2OHOP-OHOHOHCH2OOP-OHOHOH(a) (b)76 Computational analysis The canonical substrate (CDP-ME) and alternate substrate (ME-2,4-PP) was were docked at IspD active site. The distance between the phosphate of CTP and 4’-phosphate oxygen of canonical substrate MEP is 6 Å, whereas the distance between CTP and alternate substrate ME-2,4-PP is 5.6 Å (Figure 3-11). The distance between the phosphate of CTP and 2’-phosphate oxygen of ME-2,4-PP is 8.3 Å. This suggests that the addition of CMP at 4’-phosphate is unlikely. Figure 3-11 Canonical substrate (MEP) and alternate substrate (ME-2,4-PP) docked on IspD. The substrates for IspD and IspE were docked (Figure 3-12) and revealed that they bind in the same active pocket but at the different regions in the pocket. CDP-ME is the canonical substrate whereas, MEP is an alternate substrate for IspE (Figure 3-13). 77 Figure 3-12 Substrate alignments in the active site for canonical MEP pathway and bifurcation pathway. Top left image: MEP and CTP in IspE, top right image: ME-2,4-PP and CTP in IspE, bottom left image: CDP-ME and ATP in IspE, bottom right image: MEP and ATP in IspE. 78 Figure 3-13 (a) Canonical substrate (CDP-ME) and (b)alternate substrate (MEPP) docked on IspE. 3.4 Discussions The discovery and study of IspDF led me to study the MEP pathway cascade. The in vitro study conducted revealed plausible bifurcation step in the pathway. The initial bioluminescent assay showed ATP consumption without a substantial rise in ADP. To investigate ADP fate, HPLC analysis was conducted. This revealed accumulation of AMP by dephosphorylation of ADP. Reactions were conducted using ADP as the sole phosphate donor and analyzed on LC-MS. This showed that the kinase, IspE can accept ATP as well as ADP as phosphate donors to phosphorylate MEP to ME-2,4-PP. ME-2,4-PP was later confirmed by mass fragmentation pattern. The rates of this bifurcation reaction are very low compared to the canonical reaction and the conversion did not reach 100 %. Since, chemical synthesis of ME-2,4-PP was unsuccessful, the final reaction product could not be fully characterized. This study gives enough evidence to investigate the cascade of IspD, IspE and IspF in detail as future work. 79 Chapter 4: Study of catalytic promiscuity of terpene synthases 4.1 Introduction Terpenoids are secondary metabolites that form part of plants’ defense mechanisms. They also have chemical and biological significance. Terpenoids have been used as anti-infectives, anticancer, antioxidants, pesticides etc. since they are naturally tailored to bind to physiological targets (cellular components and receptors). The structure of a typical terpene comprises a hydrocarbon scaffold and has multiple chiral centers and some heteroatoms. These attributes make them lipophilic and volatile appealing their use in cosmetic, flavor and fragrance industry to a greater extent. These features also help to improve their bioavailability in natural targets. They have high calorific value because of the hydrocarbon backbone and hence are also explored as fuel additives. Such highly useful properties of the diverse class of terpenoids are the result promiscuous nature of the terminal steps of the biosynthetic pathway. These two terminal steps are catalyzed by catalytically promiscuous enzymes, terpene synthases (TSs) and P450 monooxygenases, in the order they take part in the biosynthesis. Terpene synthase acts on the products of prenyltransferase generating a pool hydrocarbon scaffolds. P450 monooxygenases then functionalize each of the scaffolds to more diverse structures. Hence, efforts of selectivity improvements towards one terpenoid product must start from addressing TS promiscuity. Terpenes occur in low concentrations in their natural hosts like plants merely due to their physiological purpose and need. Moreover, the production of terpenoids in plants is limited by several geographical and climatic factors122,123 and farming plants for terpenoid production are a threat to biodiversity. Extraction and purification of the desired terpenoid from plant’s complex matrix require arduous124–126 techniques making it uneconomical. Relying on the natural supply of 80 these trace terpenoids is non-viable at industrial scale127. For example, the prices and availability of limonene in the market are determined by cultivation in Southern American countries and are prone to fluctuations due to climatic variations and political instability. Uncertain and lower availability of isoprenoids has discouraged their explorations for applications on a large scale. The use of engineered microbial cell factories like bacteria has proved to be a more viable and sustainable option. That requires engineering the biosynthetic pathway in the heterologous host. It provides advantages like utilization of inexpensive carbon128,129, ability to improve titer by genetic manipulations81,104,130,131, faster and stable rate of production, engineering selectivity towards single product and simpler downstream processing. Some examples of developed commercial processes are- isoprene, farnesene, artemisinin and squalene from Amyris; valencene, nootkatone and sandalwood oil from DuPont; elemene and patchouli oil from Isobionics5. The inputs from the study of flux improvements from chapters 2 and 3 can be used as a modular platform strain. When doing so, it is undesired to have multiple products that not only lead to lower titers but also increase the cost of downstream purification. TS promiscuity is restrictive to the potential use of a heterologous expression as a means of production. This needs to be studied before engineering the enzymes and the pathway. Catalytic promiscuity of TSs TSs are the most diverse class of enzymes that yield a plethora of products by simple catalytic steps. The flexible polyisoprenyl diphosphate substrate (GPP, FPP, GGPP etc.) occupy the TS active site depending on the site contour. The first step of transformation involves the generation of a reactive substrate carbocation through abstraction of pyrophosphate. This step is mediated by divalent metal ion like Mg+2 and the motive DDxxD plays a role in stabilization of 81 product as well as ionic pyrophosphate group132. The carbocation then undergoes cascades of hydride shifts and/or cyclization generating diverse transition state structures (TSS). The cation is then neutralized either by proton loss or nucleophilic attack to generated terpenes. The trajectory of this cascade is determined by the stability of the substrate as well as intermediary steps. The promiscuity in the carbocation neutralization cascade determines the extent of promiscuity of the TS. Factors responsible for TS promiscuity Promiscuous nature of an enzyme is an intermediary step in the evolutionary trajectory (Figure 4-1 reproduced) of an ancient catalytically selective enzyme to achieve newer function133. This is evident from the occurrence of more than one TSs from the same class in a single species that bear high sequence similarity with each other. Phylogenetic analysis of these TSs also reveals their ancestral origin134–136. Figure 4-1 Enzyme evolution trajectory to achieve the newer function (reproduced133) 82 The TS active site molds itself on the substrate binding to more product-like orientation, for example, ‘open’ and ‘closed’ conformations of aristolochene synthase from Aspergillus terreus137. Reactions of terpene synthases are thermodynamically favorable if the active site contour is more product-like and generate a single product. Whereas, if the contour is less product-like, the TS is promiscuous and generates multiple products138. As a consequence, one or a few amino acid changes in the active site makes a huge change in product specificity 139–142. Apart from intrinsic parameters like evolution and active site structure, extrinsic parameter like enzyme environment plays a role as well. In vitro reactions of recombinant E. coli lysate expressing taxadiene synthase from Taxus brevifolia with GGPP yielded single product, taxa-4(5),11(12)-diene143. The same enzyme when expressed along with MEP pathway cassette and GGPP synthase in E. coli K12, produced a mixture of taxadiene and isotaxadiene derivatives in vivo144. The selectivity for taxadiene was reported to be 96.1 % in vivo. When the same enzyme was expressed in E. coli BL21(DE3) strain, it produced only 80 % taxadiene in vivo137. Effects of culture conditioning and heterologous host on product yield are well studied. But their influence on catalytic promiscuity is not explored systematically. Thorough knowledge of such factors contributing to the loss of flux can help to optimize the system further. Study of TS activity and promiscuity TSs fall into two main classes depending on their mechanism of generation of a carbocation from prenyl diphosphate substrates. Class I TSs involve a metal divalent aided chemistry to generate the cation and inorganic pyrophosphate. Whereas, class II TSs involve protonation of the substrate double aided by acidic residue (like aspartate) to yield a carbocation. MTSs exhibit class 83 I mechanism and the active site is located in α-helical folds145. The TSs studied here have been reported to belong to class I. Diterpene synthases from Norway spruce- isopimaradiene synthase (PaIso) and levopimaradiene/abietadiene synthase (PaLAS), exhibit very different catalytic selectivity even though they share 91 % protein sequence similarity. After homology modeling, they identified catalytic AC domain in both the TSs. When this domain was swapped, the catalytic product profiles were reversed. The specific two amino acids identified in both TSs (H694 and S721 in Palso, and Y686 and A713 PaLAS) were swapped, it did not entirely reverse the product outcome. A site-directed mutations A713D in PaLAS extinguished its native catalytic activity and produced 96% of PaIso activity. This study was able to identify four mutations that reciprocally reversed the catalytic profiles of these TSs145. This study of identification of active site structure on homology modeling, subsequent identification of residues and mutagenesis to identify the impact lead to the crucial property of TSs of catalytic plasticity. The rate-limiting step in this strategy is the generation of mutants, their characterization and the iterative process until the desired outcomes are achieved. Another example of such a study that resulted in the identification of disparity between crystal structure and catalytically active structure. The classical crystal structure reported for taxadiene synthase (TXS) is an ‘open’ structure with GGPP oriented in catalytically non-feasible conformation. This structure was reported to be catalytically non-functional and a new ‘closed’ structure was postulated based on molecular dynamic (MD) and docking studies145. The new structure verified the induced fit mechanism of involvement of R754, S713, V714 amino acid triad. The study is an excellent example of delineation of TS reaction mechanism where the discrepancy 84 with the active form of the enzyme is deduced from docking studies. The inputs for the study were provided by in vitro and in vivo TS expression. For MTSs, apart from the involvement of aspartate-rich DDxxD motif at the active site, a study revealed presence of tandem arginine (RR) motif upstream and deletion of all the amino acids upstream to it including RR motif in limonene synthase from Mentha spicata rendered the enzyme inactive towards GPP as a substrate146. This also implied that the RR motif is required for GPP to linalyl pyrophosphate (LPP) isomerization. Another study on the same enzyme reported electrostatic interactions of a first arginine residue (R58) of the RR motif with E363 residue and H-bond interactions of a second arginine residue (R59) with side chains of V357 and Y435. These interactions anchor the arginine side chains to the outside of the active site and thereby maintaining the closed active site structure147. The same study also reported that R315 forms H-bond with phosphoester oxygen of pyrophosphate after isomerization to LPP. Whereas, D496 forms the H-bond with phosphoester oxygen of pyrophosphate of GPP147. Apart from this study, a study conducted on bornyl synthase from Salvia officinalis and its crystal structure, revealed the role of R314 and R493 in forming H-bonding with the pyrophosphate and presence of aromatic residues leading to π-cation interactions stabilizing cationic structure148. Cineole synthase from Salvia fruticosa contains N338 residue that is reported to activate the water molecule for hydroxylation yielding terpenes with hydroxy groups148. Homology models of pinene synthase from Abies grandis revealed the presence of R184, R365 and C543 residues at active site149. These experiments on MTS activity were based on the enzyme crystal structure and homology model development. The use of molecular dynamics (MD) and quantum mechanics (QM) approach alone falls short in defining effective boundary conditions and results in simulations performed in a non-85 natural environment (like gas-phase model or water as solvent)150. Thought it is higher throughput, QM calculations involve a wide range of approximations and speculations are often limited by the resolution of the system and necessitates verification by wet lab experiments. Despite advancements in our understanding of these enzymes, much remains to be learned about the TS biocatalysis, especially their chemical control and how individual TSs diversified from the ancestral superfamily. Research variables and scope The variables in the study are a flux of substrate carbon, the source of the TSs and product structural diversity. I designed a controlled study where substrate carbon content and TS source were kept the same and the TSs that generated a diverse class of products were selected. I selected monoterpene synthase as a prototype as it is widely distributed among the plants and contains diverse product profiles. Monoterpenes are acyclic (hydrocarbon like myrcene and hydroxylated like linalool), monocyclic or bicyclic (with a combination of 6 and 3-membered rings in carene or a combination of 6 and 4-membered rings in pinene). The second factor that was taken into consideration is evolutionary pressure on the enzymes selected. Since enzymes isolated from a single plant have experienced similar evolutionary pressures, I selected Norway spruce (Picea abies), a coniferous tree as our model source. Isolation and characterization of MTSs are reported136,151 and were gifted to us Joerg Bohlman from his Treenomix consortia. In this work, I started developing a strategy to compare TSs in vitro and in vivo product distribution. These product profiles then guided us to dock substrates, transition states and products to deduce the catalytic mechanism. This study involves four enzymes carene synthase, myrcene synthase, 86 limonene synthase and linalool synthase and their successful expression in E. coli without protein engineering. 4.2 Materials and methods Strains and plasmid construction All strains, plasmids and genes used in this study are listed in Table 4.1. Genes dxs, ispD, ispF and idi were amplified from E. coli strain K12 genome. GPPS was amplified from pTrc-trGPPS(CO)-LS that was a gift from Jay Keasling (Addgene plasmid # 50603)152. Genes CarTS, MyrTS, LimTS and LinTS were kindly provided by Joerg Bohlmann136 and were suitably amplified. All constructs were sequenced and verified from Genewiz Inc., USA. Table 4.1 Strains, plasmids and genes Strains Description Source E. coli DH5α Cloning strain New England Biolabs (# C2987) E. coli BL21(DE3) Expression strain New England Biolabs (#C2527) E. coli BL21-CodonPlus (DE3) RIPL Expression strain Agilent Technologies (#230280) E. coli strain K12 Gene amplification Sigma-Aldrich (#EC1) SACar E. coli BL21(DE3) transformed with pSACar This study SAMyr E. coli BL21(DE3) transformed with pSAMyr This study SALin E. coli BL21(DE3) transformed with pSALin This study SALim E. coli BL21(DE3) transformed with pSACar This study SALim+ BL21-CodonPlus (DE3) transformed with pSACar This study Plasmids Description Source pSACar Ampr; GPPS and CarTS under trc promoter; dxs, ispD, ispF and idi under T7 promoter; pBR322 ori This study 87 pSAMyr Ampr; GPPS and MyrTS under trc promoter; dxs, ispD, ispF and idi under T7 promoter; pBR322 ori This study pSALim Ampr; GPPS and LimTS under trc promoter; dxs, ispD, ispF and idi under T7 promoter; pBR322 ori This study pSALin Ampr; GPPS and LinTS under trc promoter; dxs, ispD, ispF and idi under T7 promoter; pBR322 ori This study Genes Description Source dxs 1-Deoxy-D-xylulose-5-phosphate synthase NCBI Gene ID: 945060 ispD 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase NCBI Gene ID: 948269 ispF 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase NCBI Gene ID: 945057 idi Isopentenyl-diphosphate Delta-isomerase NCBI Gene ID: 949020 GPPS Geranyl pyrophosphate synthase 152 CarTS (+)-3-Carene synthase from Picea abies 136 MyrTS Myrcene synthase from Picea abies 136 LimTS (-)-Limonene synthase from Picea abies 136 LinTS (-)-Linalool synthase from Picea abies 136 Media and growth conditions E. coli DH5α was used for plasmid construction and cultivated at 37 °C in Luria Bertani (LB) media (purchased from Sigma-Aldrich). E. coli BL21(DE3) and E. coli BL21-CodonPlus (DE3) were used as expression hosts. Successful transformants of SACar, SAMyr, SALin and SALim were selected on LB agar plate containing 100 µg/mL ampicillin. Successful transformants of SALim+ were selected on LB plate containing 100 µg/mL ampicillin and 50 µg/mL chloramphenicol. For flask fermentation, a single clone was inoculated in a media containing required antibiotics; supplemented with magnesium chloride (procured from Thermo Fisher Scientific) and 88 grown at 37 °C for 6 h. The inoculum was diluted to 15 mL with the medium to OD600 of 0.2 and allowed to grow at 37 °C. Cultures were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, purchased from Sigma-Aldrich) at an OD600 of 0.8 and overlaid with dodecane to trap monoterpenes produced. Fermentations were continued at 37 °C, 30 °C, 22 °C and 16 °C. 30 mL glass tubes and 250 mL Erlenmeyer flasks were used, and the cultures were shaken at 220 rpm. Fermentations were carried out aerobically and micro-aerobically. For micro-aerobic fermentations, the tubes were capped with neoprene rubber stoppers and crimped whereas, flasks were screw-capped containing Teflon liner after the induction. Since terpenoids are volatile, the conditions were optimized initially for SACar clone. LB, modified LB (LBY; LB medium supplemented with 5 g/L yeast extract), terrific broth (TB; containing 24 g/L yeast extract, 20 g/L tryptone, 4 mL/L glycerol, 0.017 M monobasic potassium phosphate and 0.072 M dibasic potassium phosphate), 2x YT (16 g tryptone, 10 g yeast extract and 5 g sodium chloride, pH adjusted to 7) and Hi-Def Azure (HD, supplemented with 20 g/L glycerol) media were employed in fermentation. Hi-Def Azure media was purchased from Teknova and all rest media components were purchased from Sigma-Aldrich. In vitro reactions Proteins from cell pellets from 20 mL cultures were extracted and used to carry out in vitro reactions. Lysis buffer consisted of 50 mM Tris-HCl (pH 8.0), 10 % (v/v) glycerol, 0.1 % (v/v) Triton X100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM magnesium chloride and 3 units/mL DNAase. The samples were sonicated to completely lyse cells. Specific protein was purified from the lysate. Both total protein extract as well as purified protein solution were exchanged in reaction buffer by ultrafiltration and tested for in vitro activity using geranyl 89 pyrophosphate (GPP) as substrate. In vitro reactions consisted of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 % (v/v) glycerol, the purified protein, 0.2 mM GPP, and 0.4 mM MnCl2 overlaid with 1 mL of hexane136. Reactions were initiated by addition of substrate and allowed to proceed at 22 °C and terminated by vortexing. The hexane layer was sampled and analyzed on GC-MS. All chemicals used were purchased from Sigma-Aldrich. Metabolite analyses Metabolites were analyzed using GC-MS. The system consisted of PerkinElmer Clarus 680 gas chromatograph and Perking Elmer Clarus SQ8T mass spectrometer (70 eV). HP-5MS capillary column (25 m long, 0.2 mm internal diameter, 0.33 µm film thickness; Agilent Technologies) was used, with helium (1 mL/min) as the carrier gas. The oven temperature program was: 60 °C for 3 min, 5 °C/min to 100 °C, 40 °C/min to 270 °C and hold for 1 min. The injector was maintained at 250 °C. Dodecane from overlay was diluted 100 times and injected in split mode with a 20:1 split ratio and detected on total ion chromatogram. Qualitative and quantitative analyses were done by monitoring for m/z 93 which is a predominant monoterpene fragment. Peak identities were confirmed by running monoterpene standards and quantified using their respective calibration curves. Computational analysis The homology models for CarTS, MyrTS, LinTS and LimTS were generated by using I-TASSER153 using α-bisabolene synthase (PDB ID 3SAE), taxadiene synthase (PDB ID 3P5P) and 4S-limonene synthase (PDB ID 2ONH) as templates. The structures of all molecules that participate in the catalytic cycle were prepared in Marvin120. The molecules were then docked into 90 the active site of the enzymes using AutoDock Vina121. The input to the docking calculation comprised an initial set of atomic coordinates, and the program outputs putative poses of the molecule within the active site. A corresponding Gibbs free energy of binding (ΔG) was also computed for each pose. In order to identify the pose that yields the global minimum for ΔG, we selected the most stable pose and re-used its coordinates as the initial value for a subsequent docking calculation. We iterated this methodology until an unchanging pose was outputted. 91 4.3 Results Plasmid construction and protein expression GPPS (Abies grandis)104 and each of the terpene synthase were cloned in the pSASDFI operon (Section 2.2.2). Figure 4-2 are the images of SDS/PAGE gels stained with Coomassie dye. Figure 4-2 Protein expression and analysis on SDS/PAGE. (a) Gel image for GPPS expression, lane 1: total protein extract of E. coli BL21(DE3), lane 2: protein ladder, lane 3: total protein extract of engineered E. coli for GPPS uninduced, lane 4: total protein extract of engineered E. coli for GPPS with band corresponding GPPS (32.4 kDa) in the red box; (b) Gel image for (His)6-MTS (induction with 0.5 mM IPTG) purified by Ni-NTA column, lane 1: myrcene synthase induced, lane 2: carene synthase induced, lane 3: myrcene synthase uninduced, lane 4: carene synthase uninduced, lane 5: protein ladder, lane 6: limonene synthase uninduced, lane 7: linalool synthase uninduced, lane 8: limonene synthase induced, lane 9: linalool synthase induced. The bands corresponding with the specific protein are highlighted with red box. The bands corresponding with the specific protein are highlighted with red box. 92 LinTS could not be detected on SDS/PAGE when expressed in E. coli BL21(DE3) strain. When it was transformed in E. coli BL21-CodonPlus (DE3) RIPL, LinTS protein could be detected in the cell extract. Since the antibiotic resistance protein (beta-lactamase) has a size of 34.8 kDa, it is in Figure 4-2(a) lane 3. But after induction the band widens corresponding to GPPS that was also confirmed by protein-tag experiments Figure 4-3. Figure 4-3 Protein expression of (His)6-GPPS and analysis on SDS/PAGE. Lane 1: protein ladder, lane 2: purified uninduced GPPS (5 ug), lane 3: purified uninduced GPPS (10 ug), lane 4: purified induced (with 0.5 mM IPTG) GPPS (5 ug), lane 5: purified induced (with 0.5 mM IPTG) GPPS (10 ug) MTSs possess a transit peptide sequence at their N termini that localizes them to the plastid. The sequence was removed, and the remaining sequence was successfully cloned with other genes in the operon. The plasmid maps generated by Genewiz software are shown in Figure 4-4. 93 Figure 4-4 Plasmid maps for MTS expression system. (a) Myrcene synthase, (b) Linalool synthase, (c) Carene synthase, (d) Limonene synthase Study of parameters on SACar fermentations Two variables were optimized for fermentative production of terpenoids in recombinant E. coli- culture vessel and aeration. Four conditions (Figure 4-5) were tested for SACar in LBY media 94 in tubes and flasks were grown for 18 h after 1 mM IPTG induction at 30 °C with 10 %(v/v) dodecane overlay. Figure 4-5 Images of cultures (a) aerobic tube culture, (b) microaerobic tube culture, (c) aerobic flask culture, (d) microaerobic flask culture. The tube cultures produced less biomass as well as carene (Figure 4-6). Carene production was improved by 8 to 20-fold when tubes were replaced by flasks for fermentation. The biomass density was marginally lower in microaerobic flask cultures and had higher normalized carene titer (µg/L/OD). 95 Figure 4-6 Effect of culturing conditions on SACar. TA: aerobic tube culture, TM: microaerobic tube culture, FA: aerobic flask culture, FM: micro-aerobic flask culture. Primary Y-axis is carene titer and secondary Y-axis is normalized carene titer. The other culture conditions were: 30 °C temperature, LBY media (supplemented with the antibiotic and 2 mM MgCl2), 18 h incubation time and 10 % dodecane overlay. The next factor studied was incubation temperature after induction in microaerobic flask cultures (Figure 4-7). Temperatures ≤22 °C had lower OD600 but the higher carene production. At higher temperature, cells too grew faster but made lower carene. Hence 22 °C was determined to be optimal for the objective and was used for all subsequent experiments. 00.10.20.30.40.50.601234567TA TM FA FMCarene produced (µg/L/OD)Carene produced (µg/L)µg/L µg/L/OD96 Figure 4-7 Effect of incubation temperature on carene production. Primary Y-axis is carene titer and secondary Y-axis is normalized carene titer. The other culture conditions were: micro-aerobic flask cultures, LBY media (supplemented with the antibiotic and 2 mM MgCl2), 18 h incubation time and 10 % dodecane overlay. Study of strain and fermentation parameters on monoterpene production Strains SACar, SAMyr and SALin, when grown in LB media in shake flask fermentation, produced monoterpenes. Strain SALim, when grown in LB as well as other media, did not produce detectable levels of any monoterpene. pSALim transformed in E. coli BL21-CodonPlus(DE3) RIPL produced monoterpenes. E. coli BL21-CodonPlus(DE3) RIPL contains extra copies of rare tRNA genes to aid expression of heterologous proteins. Four different fermentation parameters like the role of media, divalent ion concentration, percent dodecane overlay and quantity of inducer were studied for engineered strains SACar, 00.20.40.60.811.21.41.6012345678916 22 30 37Carene produced (µg/L/OD)Carene produced (µg/L)Temperature (°C)µg/L µg/L/OD97 SAMyr, SALin and SALim+. Sequential optimization of each parameter was conducted. I had found that due to the volatile nature of products, sealed cultures maintain the most consistent environment required for the study. All these experiments were performed in micro-aerobic fermentative conditions in screw-cap shake flasks. I also observed that the biomass produced had a direct relation with monoterpene titers and each strain produced a mixture of monoterpenes. The titer of major product was monitored (carene for SACar, myrcene for SAMyr, linalool for SALin and limonene for SALim+) in each case. Though strains SACar and SALim+ gave high titers in TB, it generated way more biomass and LBY outweighed TB in when OD600 was factored in for titers of SACar and SALim+ (Figure 4-8). SAMyr consistently displayed higher myrcene production in LBY than any other media tested. Linalool titers were more than three times higher in TB medium than others. All further analyses on SACar, SAMyr and SALim+ were performed in LBY media whereas for SALin were performed in TB media. Magnesium ions were supplemented in the media optimized earlier for each strain and are required for catalytic activity of MTSs136. I observed that (Figure 4-9) Mg2+ it is required for the monoterpene production, but it did not have any effect on cell growth. 2mM magnesium chloride in the media was found to be optimum and employed in all subsequent experiments. 98 Figure 4-8 Effect of growth media on terpene production. (a) SACar, (b) SAMyr, (c) SALin, (d) SALim+. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2, 18 h incubation time and 10 % dodecane overlay. 00.10.20.300.10.20.30.40.50.60.70.80.91LB LBY 2YT TB HDLimonene produced (µg/L/OD)Limonene produced (µg/L)µg/L µg/L/OD00.40.81.21.6024681012LB LBY 2YT TB HDCarene produced (µg/L/OD)Carene produced (µg/L)µg/L µg/L/OD00.10.20.30.400.20.40.60.8LB LBY 2YT TB HDMyrcene produced (µg/L/OD)Myrcene produced (µg/L)µg/L µg/L/OD00.40.81.21.602468LB LBY 2YT TB HDLinalool produced (µg/L/OD)Linalool produced (µg/L)µg/L µg/L/OD(a) (b)(c) (d)99 Figure 4-9 Effect of Mg+2 concentration on terpene production. (a) SACar in LBY media, (b) SAMyr in LBY media, (c) SALin in TB media, (d) SALim+ in LBY media. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 18 h incubation time and 10 % dodecane overlay. 00.20.40.60.811.20123456781 2 3Linalool produced (µg/L/OD)Linalool produced (µg/L)µg/L µg/L/OD00.050.10.150.20.250.300.10.20.30.40.50.60.70.80.911 2 3Limonene produced (µg/L/OD)Limonene produced (µg/L)µg/L µg/L/OD00.050.10.150.20.2500.10.20.30.40.50.60.70.80.91 2 3Myrcene produced (µg/L/OD)Myrcene produced (µg/L)µg/L µg/L/OD00.40.81.21.6201234567891 2 3Carene produced (µg/L/OD)Carene produced (µg/L)µg/L µg/L(a) (b)(c) (d)MgCl2 concentration (mM)100 Monoterpenes are volatile and cytotoxic. In situ biphasic extraction serves the purpose of capturing monoterpenes and alleviating toxicity136. 10 % (v/v) dodecane was observed to be sufficient for the capture of monoterpenes in cultures SACar, SAMyr and SALim+. However, SALin required 30 % (v/v) overlay with dodecane (Figure 4-10). This optimized dodecane overlay was used in subsequent experiments. 101 Figure 4-10 Effect of dodecane overlay on terpene production. (a) SACar in LBY media, (b) SAMyr in LBY media, (c) SALin in TB media, (d) SALim+ in LBY media. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2 and 18 h incubation time. 00.40.81.21.6201234567895 10 20 30Carene produced (µg/L/OD)Carene produced (µg/L)µg/L µg/L/OD00.040.080.120.160.200.10.20.30.40.50.60.70.80.95 10 15 20Myrcene produced (µg/L/OD)Myrcene produced (µg/L)µg/L µg/L/OD% Dodecane overlay (v/v)00.30.60.91.201234567895 10 20 30 40Linalool produced (µg/L/OD)Linalool produced (µg/L)µg/L µg/L/OD00.050.10.150.20.2500.10.20.30.40.50.60.70.80.915 10 15 20Limonene produced (µg/L/OD)Limonene produced (µg/L)µg/L µg/L/OD(a) (b)(c) (d)102 Induction with IPTG was the next step in optimization. Both MEP pathway operon and terpene biosynthesis operon were cloned under IPTG inducible promoters. The strains were observed to require differing amounts of IPTG for optimal induction (Figure 4-11). Optimal concentrations of IPTG for strains were: 0.7 mM for SACar, 0.25 mM for SAMyr, 0.2 mM for SALim+ and 0.1 mM for SALin. Higher concentration of IPTG had a negative effect on cell growth and subsequent monoterpene production which is consistent with the literature. This effect was pronounced for SALim+ cultures. 103 Figure 4-11 Effect of IPTG induction on terpene production. (a) SACar, (b) SAMyr and (d) SALim+ in LBY media with 10 % dodecane overlay; (c) SALin in TB media with 30 % dodecane overlay. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. The fermentation conditions were: micro-aerobic flask cultures, 2 mM MgCl2 and 18 h incubation time. IPTG concentration (mM)00.050.10.150.20.2500.10.20.30.40.50.60.70.80.900.10.15 0.20.25 0.30.50.75 1Myrcene produced (µg/L/OD)Myrcene produced (µg/L)µg/L µg/L/OD00.050.10.150.20.250.30.3500.10.20.30.40.50.60.70.80.9100.10.20.30.40.50.75 1Limonene produced (mg/L/OD)Limonene produced (mg/L)µg/L µg/L/OD00.30.60.91.201234567800.0250.050.0750.10.20.30.40.5Linalool produced (µg/L/OD)Linalool produced (µg/L)µg/L µg/L/OD00.40.81.21.6202468101200.10.25 0.50.60.70.80.9 1Carene produced (µg/L/OD)Carene produced (µg/L)µg/L µg/L/OD(a) (b)(c) (d)104 Table 4.2 summarizes the fermentation conditions that were found to give higher terpenoid titers from selected sample space. Table 4.2 Summary of fermentation conditions selected Strain Growth media Magnesium chloride concentration (mM) IPTG concentration (mM) Dodecane overlay (% v/v) SACar LBY 2 0.70 10 SAMyr LBY 2 0.25 10 SALim LBY 2 0.20 10 SALin+ TB 2 0.10 30 Aerobic and micro-aerobic fermentations All shake flask fermentations were run using optimized parameters. SACar, SAMyr and SALim+ fermentations were run in LBY media supplemented with 2 mM MgCl2, overlaid with 10 % (v/v) dodecane. SACar was induced with 0.7 mM IPTG, SAMyr with 0.25 mM IPTG and SALim+ with 0.2 mM IPTG. SALin fermentations were run in TB media supplemented with 2 mM MgCl2, overlaid with 30 % (v/v) dodecane on induction with 0.1 mM IPTG. The conditions of higher titers implied that the flux through the pathway is high enough that the limiting step is the last step catalyzed by TS. Figure 4-12 shows the major monoterpene product produced (carene for SACar, myrcene for SAMyr, linalool for SALin and limonene for SALim). SACar, SAMyr and SALin aerobic fermentations gave higher titers of major monoterpene product than micro-aerobic conditions. Whereas, SALim+ performed better in oxygen-limited conditions. All four strains had a similar trend in dependence of monoterpene production on fermentation time. The monoterpene titers 105 increased with time upon induction until a point where it reached a maximum and stayed constant. All four strains took 18 h to 26 h to reach the saturation point. Because of the volatile nature of terpenoids, later drop in titer can be attributed to loss from the system by evaporation. 106 Figure 4-12 Fermentative production of monoterpenes over time. (a) SACar, (b) SAMyr, (c) SALin, (d) SALim+. Primary Y-axis is terpene titer and secondary Y-axis is normalized terpene titer. Fermentation conditions are mentioned in Table 4.2. 02460510150 10 20 30 40 50 60 70 80CarTS00.20.40.60.800.20.40.60.810 10 20 30 40 50 60 70 80MyrTS04812160369120 10 20 30 40 50 60 70 80LinTSMajor monoterpene produced (µg/L) Major monoterpene produced (µg/L/OD) 00.10.20.30.40.00.30.60.91.20 10 20 30 40 50 60 70 80LimTSµg/L Aerobic µg/L Micro-aerobic µg/L/OD Aerobic µg/L/OD Micro-aerobic(a)(b)(c)(d)107 Maximum carene production for SACar in aerobic conditions was 12.6 µg/L at 21 h whereas in micro-aerobic, it was 10.3 µg/L carene at the end of 26 h. Myrcene titers in SAMyr for aerobic conditions reached a maximum of 0.84 µg/L at 26 h but 0.75 µg/L at 21 h for micro-aerobic fermentations. SALim+ titers for limonene in aerobic conditions (0.93 µg/L) were less than micro-aerobic conditions (1.08 µg/L) both reaching maxima at 26 h after induction. SALin fermentations were greatly influenced by oxygen. I observed a 30% difference in the maximum linalool concentration (aerobic: 10.3 µg/L at 18 h and micro-aerobic: 6.9 µg/L at 21 h). Except for SALim+ cultures, all other tree had higher monoterpene yield in aerobic conditions than micro-aerobic. So, I tested limonene synthase from Mentha spicata104 expression in E. coli BL21(DE3) and limonene production in aerobic and micro-aerobic fermentations. Aerobic cultures produced limonene at higher concentrations initially and after the third day of fermentation, micro-aerobic surpassed aerobic titers. I monitored these fermentations for five days and the maximum titer was reached on the fourth day (5 mg/L). Cell growth during fermentation was monitored as optical density at 600 nm. LBY fermentations reached maximum OD600 in the range of 6 to 8. Whereas, TB cultures reached a higher OD600 of 12-14. For all, OD600 did not change substantially after the third day and was higher for aerobic cultures than micro-aerobic except SACar. SALim+ cultures showed a drop in OD600 after the fourth day due to cell death and lysis. Metabolite analyses Dodecane samples from fermentations and hexane samples from in vitro reactions were analyzed on GC-MS. Chromatograms were analyzed in SIM mode with m/z = 93, a characteristic fragment for monoterpenes. In vitro product profiles for both total protein extract and purified 108 protein extract were similar to each other as well as literature140,129. I found some differences not only with the amount of a specific product being made but also which product is made. I identified newer products of the TSs than what had been reported previously and were dependent on the supply of oxygen during fermentation. Qualitative and quantitative product profiles are listed in Table 4.3 for nine different monoterpenes that were produced by the strains in varying concentration. Percent share of a terpene out of total was calculated on molarity basis with standard error for triplicates. Products of these monoterpenes are hydrocarbons (C10H16) except linalool (C10H18O) that has a hydroxyl group. The product distribution is enriched in different classes of monoterpenes encompassing acyclic (myrcene, ocimene, linalool), monocyclic (limonene, terpinene, terpinolene) and bicyclic (carene, pinene, sabinene). Table 4.3 Monoterpene product distribution and percent total Products Conditions SACar aerobic fermentations (This study) SACar micro-aerobic fermentations (This study) CarTS in vitro reactions (This study) Previously reported CarTS in vitro reactions151 Carene 83.4±0.9 76.7±0.8 80.1±0.6 78.0 Terpinolene 3.9±0.2 4.8±0.2 9.9±0.4 11.0 Sabinene 8.5±0.2 10.4±0.4 4.3±0.2 5.0 Myrcene 2.5±0.2 2.6±0.2 2.8±0.3 3.0 Limonene 1.2±0.2 2.2±0.2 0.1±0.1 0.0 Terpinene 0.3±0.2 0.8±0.3 1.5±0.3 1.6 Pinene 0.1±0.1 2.5±0.4 0.7±0.3 0.9 Phellandrene 0.0 0.0 0.6±0.2 0.7 109 SAMyr aerobic fermentations (This study) SAMyr micro-aerobic fermentations (This study) MyrTS in vitro reactions (This study) Previously reported MyrTS in vitro reactions129 Myrcene 98.7±0.8 93.7±0.7 100 100 Carene 1.0±0.2 6.1±0.5 0 0.0 Pinene 0.3±0.1 0.3±0.1 0 0.0 SALim+ aerobic fermentations (This study) SALim+ micro-aerobic fermentations (This study) LimTS in vitro reactions (This study) Previously reported LimTS in vitro reactions129 Limonene 92.1±0.6 88.7±0.5 90±0.6 89.9 Myrcene 5.9±0.1 8.2±0.3 5±0.2 5.2 Pinene 0.8±0.1 1.0±0.1 5±0.2 4.9 Linalool 1.2±0.3 0.5±0.2 0 0.0 Carene 0.4±0.2 1.6±0.2 0 0.0 SALin aerobic fermentations (This study) SALin micro-aerobic fermentations (This study) LinTS in vitro reactions (This study) Previously reported LinTS in vitro reactions129 Linalool 97.0±0.9 79.8±0.8 99.2±0.9 98.2 Ocimene 2.8±0.5 5.4±0.3 0.8±0.2 1.0 Carene 0.1±0.2 14.6±0.4 0 0.2 Myrcene 0.0 0.0 0 0.2 Terpinolene 0.0 0.0 0 0.1 Pinene 0.0 0.2±0.2 0 0.0 Others - - - Each <0.07 Study of terpene synthase catalytic mechanisms The reaction mechanisms for monoterpene production from GPP are depicted in Figure 4-14. It originates with the abstraction of the pyrophosphate group to generate primary geranyl carbocation. This carbocation can exist in two isomeric conformations: cisoid and transoid. For 110 the cyclic monoterpenes to form, the geranyl carbocation undergoes 6,1-ring closure to generate α-terpinyl carbocation. The distance between the cation and C6 in trans isomer is 5.8 Å whereas, in cis isomer it is 4.7 Å (Figure 4-13 (a) and (b)). Hence, cyclization is favorable in cisoid conformation than in transoid. α-Terpinyl cation then undergoes a range of cyclization, rearrangements and hydride shifts before the reaction is terminated by deprotonation. Similarly, the formation of α-terpinyl carbocation is favorable than β-terpinyl carbocation (Figure 4-13 (c) and (d)). Figure 4-13 Isomeric structures of carbocations. (a) Cisoid geranyl carbocation, (b) transoid geranyl carbocation, (c) α-terpinyl carbocation and (d) β-terpinyl carbocation. 111 Figure 4-14 Reaction mechanisms of monoterpene synthases. Monoterpene structures’ color is based on its penultimate carbocation origin. The α-terpinyl carbocation leads to an array of cyclic monoterpenes through rearrangements. Carene, limonene and terpinolene are deprotonation products of the α-terpinyl carbocation. Carene synthesis involves 1,7-closure to generate C3 ring. α-Terpinyl carbocation can Geranyl pyrophosphate Primary geranylcation (cisoid)Tertiary geranyl cationLinaloolMyrceneα-Terpinyl cation Terpenen-4-yl cationPinyl cationPinene Thujyl cationSabineneγ-TerpineneTerpinoleneLimoneneCarene6,1-closure2,7-closure 6,7-hydride shift 2,6-closureH2O1234567112 undergo 2,7-closure to form a pinyl cation containing C4 ring that then forms pinene. α-Terpinyl carbocation can either form γ-terpinene through neutralization or can undergo a 6,7-hydride shift to thujyl carbocation containing C3 ring. Thujyl cation then generates sabinene by deprotonation. Formation of acyclic monoterpenes like myrcene and linalool proceeds via the rearrangement of cisoid primary geranyl carbocation to form a tertiary carbocation. The tertiary carbocation is then neutralized to form myrcene by deprotonation and to linalool by water addition. Following two characteristics were studied to evaluate the plausible catalytic cascade for each terpene synthase – the amino acid located at the active site and their proximity to the reactive centers; binding energetics of the transition state structure (TSS). It is reported that amino acids like arginine, histidine, tyrosine, aspartate, glutamate are involved in hydrogen-mediated mechanisms17,141,155. The orientation of TSSs and products were docked on TS active site and amino acid residues in 5.0 Å radius were identified. Following discussion is with a focus of these docking experiments. PyMOL software was employed to study the results and generate the images. Cyclization of primary geranyl carbocation (cisoid) to the α-terpinyl carbocation The likelihood of this reaction decides whether the terminal products are acyclic or cyclic. For the cyclization to happen, the C6 comes closer to the cationic carbon decreasing the angles between C3, C4, C5 and C6 (Figure 4-15). For all the docking results, the active site surface is highlighted in grey. 113 Figure 4-15 Changes in the orientation of primary geranyl cation on 6,1-closure to form the α-terpinyl cation. (a) Orientation of primary geranyl cation, (b) orientation of α-terpinyl cation. The TSSs are highlighted in green. Figure 4-16 shows the outcomes of docking the geranyl carbocation at the active site. CarTS active site contains R480, -NH2+ group of which is at a 4.1 Å from the geranyl carbocation. The arginine is reported to catalyze pyrophosphate group removal and catalyze proton elimination-condensation cascade155,156. Moreover, -NH+ group of H436 and -SH group of C236 are positioned at the distances of 4.3 Å each from the C6 that trigger π-electron shift and cyclization. Moreover, the energy of binding to CarTS is -4.7 kcal/mol. Similarly, LimTS contains -NH2+ group of R303 residue at 3.6 Å, R485 residue at 4.2 Å and R301 residue at 4.9 Å from the cationic carbon. The C6 atom is also oriented at 4.5 Å from R479. The binding energy of geranyl carbocation is -5.0 kcal/mol due to the arginine triad (R301, R303 and R485). Both these enzymes have an aromatic amino acid residue- Y561 in CarTS and F566 in LimTS close to methyl groups of C7, but they are oriented away from the direction of cyclization. 114 Figure 4-16 Role of amino acid residues in TS active site towards cisoid primary geranyl cation reactivity. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in green, the favorable interactions for 1,6-closure are highlighted in green dotted lines and unfavorable interactions are highlighted in purple dotted lines with distance measurements. 115 MyrTS and LinTS product spectra involve the majority of acyclic terpenoids. When the geranyl carbocation was docked in their active sites, few/no arginine residues were observed in 5.0 Å radius. MyrTS has R482 whose -NH2+ is at 4.0 Å from the carbocation and Y410 whose hydroxy group is at 4.2 Å from C6. But the benzene ring of F563 is at 3.7 Å and 4 Å from the methyl groups on C7. The ring faces the same side as the possible trajectory of the cyclization. The binding energy of primary geranyl cation in MyrTS was found to be -4.9 kcal/mol. The LinTS active site is devoid of the arginine residues, instead have Y399, Y403 and D328 whose hydroxy groups are at 3.5 Å, 3.9 Å and 4.0 Å respectively. The geranyl moiety in LinTS is surrounded by four phenyl alanine residues F317, F429, F546 and F556 in 5.0 Å radius obstructing cyclization and exhibited the highest binding energy of -5.6 kcal/mol. Formation of carene from the α-terpinyl carbocation CarTS fermentations made 77-83 % carene from the mixture, whereas the other three TSs studies made carene at less than 6 % of the mixture. This drastic difference in the product ratios is a result of the reactivity of α-terpinyl carbocation. Hence CarTS and MyrTS active sites were docked with both α-terpinyl cation and carene; and LinTS and LimTS were docked for TSS α-terpinyl cation Figure 4-17. α-Terpinyl cation TSS is dynamic inside the CarTS active site and flips on cyclization. This could result because of the location of -OH of D333 and -NH2+ of R296 at 3.8 Å and 4.6 Å respectively from C7 of the α-terpinyl carbocation. Apart from this, -SH group of C326 and -NH+ of H436 occupy active site at 4.4 Å and 4.6 Å distance from carene C7. Apart from R480 identified earlier, R488 is also present aligned with the plane of the α-terpinyl cation. CarTS is the only 116 enzyme that has cysteine residue (C326) in the catalytic site and its interaction with the terpinyl cation can lead to the cyclization to carene. MyrTS does not contain the arginine located in 5.0 Å radius. Benzene ring of F563 is at 3.3 Å from terpinyl cation and at 5.3 Å from C1 of terpinyl structure. In the case of LimTs, R485 is at 5.2 Å from the terpinyl cationic carbon. Moreover, LimTS active site also possesses cysteine residue C486 but is at 8.0 Å from the C7 of the α-terpinyl carbocation. Hydroxy group of D338 residue in LimTS active site is located at 3.7 Å from C1 generating the possibility of cyclization. The LinTS site is dominated by aromatic residues like phenyl alanine and tyrosine. Hydroxy group of T325 is at 3.8 Å from the cationic carbon. These interactions are very weak and are reflected in lower titers of carene by LinTS aerobic fermentation. The binding energies of terpinyl cation at CarTS site is -5.0 kcal/mol, at LimTS site is -5.3 kcal/mol and at MyrTs site is -5.5 kcal/mol. But at LinTS site it is -6.3 kcal/mol also suggesting a lower activity for further catalytic reactions. 117 Figure 4-17 TS active site contour towards formations of carene from the α-terpinyl cation. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in purple and the α-terpinyl cation is highlighted in green.The α-terpinyl cation interactions are highlighted in purple dotted lines and carene interactions are highlighted in green dotted lines with distance measurements. 118 Pinyl cation formation by 2,7-closure of the α-terpinyl carbocation Pinene was detected in trace levels during CarTS and LimTS fermentations. Hence, the TSSs were docked in their catalytic sites (Figure 4-18). In case of CarTS, apart from the interactions of residues with α-terpinyl carbocation in previous section 4.3.8, C2 of α-terpinyl TSS does not have any residue within 5.0 Å radius. Benzene ring of F566 located at 3.7 Å from C2 of terpinyl hinders the 2,7-closure in LimTS. And R301 and R303 are at the distance of 4.1 Å and 4.7 Å from C2 terpinyl leading the plausible catalytic activity towards pinyl cation. But in both cases, the interactions are weaker. CarTS on the other hand has dominant 1,7-closure leading to carene. Figure 4-18 TS active site contour towards formations of pinyl cation from the α-terpinyl cation. The TS is identified in the left top corner of each box. The α-terpinyl cation is highlighted in purple and the pinyl cation is highlighted in green. The α-terpinyl cation interactions are highlighted in purple dotted lines with distance measurements. 119 Acyclic terpenoid mechanisms from cisoid primary geranyl carbocation The acyclic monoterpenes such as myrcene and linalool originate from tertiary geranyl carbocation. Hence, the likelihood of formation of acyclic monoterpene products is determined by the conversion of cisoid primary geranyl cation to tertiary geranyl (Figure 4-19). In CarTS, as discussed earlier, primary geranyl cation distance from R296 and R480 is 4.6 Å 4.4 Å respectively. But the tertiary geranyl cation orientation increases the distance to 4.8 Å from R480 and >5.0 Å for R296 reducing the stability. In the case of LimTS, the primary geranyl cation is in close proximity to R301 (4.9 Å), R303 3.6 Å) and R485 (4.2 Å). Moreover, -SH of C486 is at 4.9 Å from the primary carbocation. But only hydroxy groups of D330 and Y413 are at 3.6 Å and 4.0 Å from the tertiary carbocation. This explains the higher distribution of cyclic monoterpenes for these TSs. For MyrTS, the -NH2+ group of R482 is at 4.1 Å from primary cation but 4.0 Å from tertiary cation. Moreover, R298 and D335 are at 4.4 Å and 4.6 Å respectively from the tertiary cation. Whereas in the case of LinTS, both TSSs exhibit weaker interactions and the active site is enriched in aromatic residues like Y403 and Y399. Aspartate residue D328 is at 4.5 Å from the tertiary geranyl carbocation. 120 Figure 4-19 TS active site contour towards formations of tertiary geranyl cation from cisoid primary geranyl cation. The TS is identified in the left top corner of each box. The cisoid primary geranyl cation is highlighted in purple and the tertiary geranyl cation is highlighted in green.The primary geranyl cation interactions are highlighted in purple dotted lines and tertiary geranyl cation interactions are highlighted in green dotted lines with distance measurements. 121 4.4 Discussions Optimization of fermentation conditions Mixing is an important factor in terpenoid production in cell cultures. Mixing ensures a homogeneous environment of nutrients, oxygen and metabolites; and also influences terpene extraction in the organic phase. Terpenoids are hydrophobic and are effluxed out of the cell after synthesis. They evaporate from the media due to their volatile nature. Organic solvent overlay traps the terpenoids. Tube cultures have a smaller exposed surface area and hence terpenoids can be efficiently stripped into dodecane. But the same feature hinders mixing as the dissolution of oxygen in media which was evident from low cell density. Aerobic cultures in tubes had higher cell growth but low carene detected which could be due to a loss in the environment. Flasks reached twice the OD600 than tubes. Aeration did not influence flask cultures to the extent tube cultures did towards carene production. Cultures in the flasks occupied 6 % of volumetric space which was multiple folds lower than that of tubes (50 %). Optimization of shake flask fermentations Monoterpenes are volatile and are sparingly soluble in aqueous media but have high octanol:water partition coefficient. Dodecane overlay ensured that the monoterpenes partitioned into the overlay when produced and reduced further evaporation. I carried out shake flask fermentations in the absence of dodecane overlay and extracted monoterpenes at the end of fermentation by liquid-liquid extraction with hexane, I did not detect any monoterpenes. This suggested to us that dodecane was efficient in trapping terpenes as well as reducing their cytotoxicity. Even though the reduced evaporation rates aided more terpene capture and detection, fermentation parameter optimizations were done in micro-aerobic conditions to minimize errors. 122 Another key parameter that played a vital role in improving titers was media composition. Terpene production required enriched media. This was achieved by supplementing LB media with 0.5 % (w/v) yeast extract. For SACar, SAMyr and SALim+, this substantially increased monoterpene titers. For strains expressing cyclic monoterpene as major products (SACar and SALim+), TB yielded 16 % and 23 % higher titers for SACar and SALim+ respectively than LBY by supporting higher biomass generation. It was observed that monoterpene titers in TB did not increase in comparison to the cell growth and when normalized with OD600 (µg/L/OD); and they dropped by 40 % for SACar and 30 % for SALim+. Hence LBY media was selected for these three strains. Linalool production by SALin displayed a different trend. It had higher titers as well as OD600 normalized titers in TB than any other media. pH of SALin cultures in LBY media was 7.8 and in TB media was 7.2 when measured after overnight incubation. TB media is buffered with mono and dibasic potassium phosphates. Since, linalool is oxygenated (-OH) terpene and the hydroxyl functionality is weakly acidic, higher titers in TB indicate that the buffered conditions had a positive impact. LB, LBY and 2YT are unbuffered; and yielded titers and cell densities in a similar range. HD media is buffered too but is not as enriched as TB and did not support very high cell growth throughout and hence lower linalool titers. Effect of pH on the biosynthesis of monoterpenes in E. coli is not researched much. It is reported that acidic pH had an unfavorable effect on bioconversion of limonene to perillic acid in yeast157. Another study showed the effect of pH on sesquiterpene production in E. coli and its dependence on terpenes from the same class but the different structure157. Apart from structural variability, linalool had differences in physical parameters than other monoterpenes. It has lower reported vapor pressure (0.159 mm Hg), and lower (2.97) octanol:water partition coefficient and very high solubility in water (1590 mg/L)158 (Table 4.4). This also explains the need for using higher dodecane overlay (30 % v/v) to capture 123 linalool produced. After analyzing these parameters, it is evident that the ionization state of linalool affects the partitioning, cytotoxicity and in turn its overall titers. Table 4.4 Physical properties of monoterpenes Structure Compound Log Po/w Vapor pressure (mm Hg) Solubility in water (mg/L) Myrcene 4.33 2.09 5.60 Carene 4.38 3.48 6.10 Limonene 4.57 1.55 0.65 Linalool 2.97 0.16 1590.00 124 Product profile characterization To compare the product profiles I referred to catalytic mechanisms of monoterpene biosynthesis from GPP159. Monoterpene biosynthesis involves three major cationic intermediates generated by an attack by Mg+2 leading to pyrophosphate group abstraction. These three carbocationic stages are a trans-geranyl cation, cis-linalyl cation and α-terpinyl cation; occurring in the order in the cascade of electron transfers to stabilize the ion. Product profiles of in vitro reactions performed in this study at 22 °C were similar both qualitatively and quantitatively with the one reported in the literature that were performed at 30 °C (table 2). This suggests that the strain engineering has not affected protein folding or activity. Even lower temperature for reaction incubation had no noticeable effect on product profile. Shake flask fermentation data provided us with two comparisons. First, the comparison between aerobic and micro-aerobic product profiles and second, between in vivo expressions to in vitro reactions. Discrepancies between in vitro product profiles of CarTS has been mentioned before18,151. Reiling et al. could not detect minor products of CarTS when expressed in vivo but have reported in vitro product distribution by comparing it with carene’s titer. In addition to products reported by Faldt et al., Reiling et al. detected limonene. I too observed limonene in SACar fermentations but phellandrene remained undetected in our study. The percent of carene in the products was lower; and pinene and sabinene were higher in micro-aerobic than aerobic fermentation. All these are cyclic monoterpenes originating by neutralization of α-terpinyl cation by hydride shifts. Pinene synthesis occurs from pinyl cation by 2,7-closure of the α-terpinyl cation. Sabinene is formed by 2,6-closure through thujyl cation. The absence of phellandrene in vivo suggests that phellandryl cation chemistry does not proceed in our cultures. 125 Catalytic promiscuity of MyrTS was detected in vivo with myrcene as a major product but not in vitro. Catalytic mechanisms myrcene biosynthesis involves quenching geranyl (trans) or linalyl (cis) carbocations by proton abstraction. Presence of carene and pinene in fermentation suggests stabilization of α-terpinyl cation by 6,1-closure of linalyl cation. Though pinene titers did not change substantially, carene titers were higher in the micro-aerobic fermentations. SALim+ cultures showed a similar trend. In vivo promiscuity was higher and; carene and linalool were detected as additional products apart from limonene, myrcene and pinene. In this case, linalool titer was higher in the aerobic whereas myrcene and carene titers were higher in the micro-aerobic conditions. Out of the five products, linalool formation involves water as a nucleophile and based on results, this is favored in the aerobic conditions. Fermentative levels of pinene were low in SALim+ cultures than in vitro reactions. SALin in vitro reactions detected linalool as major product and ocimene as a minor product. Other monoterpenes reported129 remained undetected in our enzymatic reactions. Micro-aerobic titer for linalool was substantially lower and carene was higher. Linalool and carene involve biosynthetic mechanisms through two completely different mechanisms as well as carbocationic stages. Bicyclic monoterpene pinene was also detected in trace levels. This suggests that the cyclization mechanisms prevailed in micro-aerobic conditions. I did not detect any acyclic or monocyclic monoterpene produced by SALin. In conclusions, I observed that linalool production was favored in aerobic conditions whereas, bicyclic monoterpene (carene and pinene) production was enhanced in oxygen-limited condition. 126 Computational analyses The homology models generated for TSs and docking various TSSs and products revealed the structural basis for TS promiscuity. All four terpene synthases contain an aspartate residue (highlighted in green in Figure 4-21) that is the first residue of DDxxD motif. This is Mg+2 ion binding motif is required for the generation of primary geranyl cation from GPP. Both CarTS and LimTS have active site contour favoring cyclization of primary geranyl carbocation to terpinyl carbocation. The residues interacting for conversion of primary geranyl carbocation to tertiary geranyl carbocation are either absent or sparsely located from the active center. Whereas, MyrTS and LinTS lack the residues at active site responsible for 6,1-closure in primary geranyl carbocation but display the active site shape favoring tertiary carbocation generation. The residues with charged side chains like arginine, histidine, aspartate and tyrosine populate the active site and perform two functions: charge delocalization and cation stabilization through solvation. The amino acids with aromatic side chain like phenyl alanine and tyrosine are enriched in the MyrTS and LinTS reducing the mobility of the TSSs for cyclization mechanisms. The residues involved at the active site are shown in Figure 4-20 and the discussion is divided for each MTS in the following paragraphs. CarTS: Residues R480, H436 and C326 are involved in 1,6-closure mechanism to generate and stabilize α-terpinyl cation. These steps facilitate the movement of the terpinyl cation and bring it in the proximity of H436, C326, D333, R296 and R488 that trigger the 7,1-closure leading to carene formation. Residue H436 and C326 are closer to C6 of the terpinyl cation, generating a possibility of 2,7-closure leading to pinene as a product. The active site also contains Y561, but the benzene ring is located away from the active site interacting minimally. 127 LimTS: Primary geranyl cation is less reactive towards the formation of tertiary geranyl cation as residues R303, R485, R301and C486 are in closer to the primary cation. Moreover, R479 in the vicinity of C6 triggers the terpinyl cation formation which then follows 2,7-closure to yield pinyl cation due to residue R303 and R301. The active site also contains aromatic residue F566 but is pointing away from the active site pocket. MyrTS: This active site contains R298, R482 and D335 that are in the closer to tertiary geranyl cation than primary geranyl cation. The proximity of C6 to Y410 could translate into cyclic monoterpene formation but juxtaposed benzene ring of F563 limits the movement of primary geranyl cation towards cyclization mechanisms. LinTS: Like MyrT, LinTS active site contains Y399, Y403, T325 and D328 that form a basket around the tertiary carbocation. Aromatic residues F317, F429, F546 and F556 surround the C1, C6 and C7 methyl groups strongly obstructing movement for cyclization. 128 Figure 4-20 TS active site contour and participating amino acid residues. The TS is identified in the left top corner of each box. The surface is highlighted in grey. 129 Comparison of product profile with computational analysis All four TSs studied belong to Picea abies and were considered to have evolved from the common ancestral enzyme. When the product spectra were compared with the outcomes of computational docking, the link between the conserved amino acid and catalytic function could be established. This comparison is summarized in Figure 4-21. Figure 4-21 Predictions for roles of the amino acid residues present in TS active site and structure-function correlations CarTS LimTS MyrTS LinTSR296 R301 R298 F317 Generation of cyclic productsC326 R303 T325 Generation of primary geranyl cationD333 D338 D335 D328 Interaction (non-cyclization) with tertiary geranyl cationH436 Y410 Y399 Interaction (cyclization) with tertiary geranyl cationY403 Stearic hindrance to α-terpinyl cation generationR480 R479 R482 Unknown but involvement in bicyclic product formationR488 R485Y561 C486F566 F563 F429F546F556130 Chapter 5: Designing an efficient bioprocess for terpenoid recovery 5.1 Introduction In my work on terpenoid biosynthesis in recombinant strains, I addressed the issues of low yields of terpenoids in a heterologous host. I studied strategies to improve flux through the MEP pathway by employing enzyme fusions. This shed light on the plausible bifurcation step in the pathway. In any case, despite the flux through engineered MEP pathway being very high, it gets attenuated further due to the promiscuous nature of the biosynthetic enzymes. My strategy of studying the mechanistic basis of terpene synthase promiscuity could be applied towards addressing low yields. However, these insights need to be translated to the chemical industry and all the efforts for improving heterologous terpenoid production will be in vain if large scale bioprocess development is not economically viable. This challenge will be addressed in the current chapter. Challenges in terpenoid bioprocess development Terpenoids are secondary metabolites that are commonly secreted out of the cell. Despite many of their economically interesting applications and advancements in the synthetic biology tools that have enabled their production in simpler hosts like bacteria and fungi, there are very few examples of their commercial production. The major challenges in bioprocess development are physicochemical and biochemical properties of terpenoids. They are volatile hydrocarbons and are cytotoxic once they exceed a certain concentration. Microbial bioprocess development is hence challenging and calls for continuous product recovery160. 131 Continuous ex situ recovery (CESR) The physicochemical properties of terpenoids (hydrophobic and volatile) are exactly opposite to the environment that they are produced in (aqueous and less volatile). These differences can be utilized to efficiently design the bioprocess. There are various techniques to efficiently capture the products that either come directly in contact with the culture media (in situ product recovery) or that run parallel to the bioreactor capturing volatile fraction (ex situ product recovery). Both these methods either involve heterogeneous liquid phase or gas phase160. The continuous extraction in the heterogeneous phase also minimizes the cytotoxicity of terpenoids. There are many examples of the development of such processes for cell-free biocatalysis16 or extraction from the cells post bioprocess161. Some examples that involve whole cell fermentation are centered towards single reaction conversion like interface bioreactor for racemic mixture resolution162. De novo bioprocesses for terpenoids widely use an organic solvent that is overlaid on the fermentation media to capture products. It employs high boiling solvents like dodecane28,152,163, isopropyl myristate164, methyl oleate165. Most of these processes are either only in experimental stages or are for high value-low volume pharmaceuticals like taxol. Drawbacks of state of the art Employment of high boiling organic phase for terpenoid recovery is efficient but not sustainable. Boiling points of these solvents are: dodecane (216 °C), isopropyl myristate (167 °C) and methyl oleate (351 °C). Whereas terpenoids are high boiling as well for example monoterpenes (>140 °C), sesquiterpene (>220 °C) etc. Hence, recovering the terpenoids back from the organic phase is an energy-intensive process often requiring distillation. Such a process loses its industrial 132 applicability and viability at product recovery-purification stage. Low boiling and recyclable solvents like hexanes, methanol, ethanol etc. are cytotoxic and cannot be used. The objective of the study Development of an efficient recovery technique for terpenoids will not only make the bioprocess economical but also enable large scale applications of terpenoids as bulk and commodity chemicals. The objective of this study is to devise a strategy that will not only be efficient, scalable and economical. The initial study was done on SACar strain as a prototype and now has become a comprehensive project lead by Azin Amiri for her PhD studying SAMyr, SALim and SALin. I used the volatile nature of these terpenoids to capture it from the exhaust gases using the adsorption mechanism. 5.2 Materials and methods Strains and fermentation conditions SACar (described in Chapter 4: page number 86) fermentative process was optimized for carene recovery in a 2 L bioreactor with 1 L working volume. Fermentation was carried out in LBY media supplemented with the appropriate antibiotic and 2 mM MgCl2. Starter cultures were grown in Erlenmeyer flask at 30 °C overnight. Production cultures in the bioreactor were inoculated with 1 % (v/v) of starter cultures and grown 37 °C. The temperature was reduced to 22 °C when the culture reached an OD600 of 0.8. When the culture temperature reached 22 °C, IPTG was added to the final concentration of 0.7 mM and fermentation was continued for 18 h. 133 Bioreactor set up Fermentations were carried out in New Brunswick Scientific BioFlo 110 equipped with probes for pH, DO, temperature; and controllers for temperature and pH. Rushton turbine system with two impellers was used for agitation with sparger located at the base of the bottom impeller. The reactor vessel was 8.5" tall and 4.92" in diameter with an internal cooling coil and an external heating blanket. pH was monitored but not controlled. Antifoam addition was not required. CESR for terpenoid The device (fluidized bed capture device, FBCD) was constructed for CESR that contained resin bed fluidized in water and was connected to the air outlet stream through a condenser. The FBCD schematic is shown in Figure 5-1. The device was made from polylactic acid (PLA) polymer with an internal diameter of 1.5 cm and a height of 4.5 cm with a capacity of 8 mL. It was covered with a mesh made of stainless steel and was filled wet resin and the inlet was connected to the air flow outlet of the bioreactor. Figure 5-1 Schematic for construction of fluidized bed capture device (FBCD) The CESR schematic is shown in Figure 5-2. Condenser prevents water loss and maintains the volume of the culture. The exhaust air enriched in evaporated terpenes then passes through the 134 fluidized bed of resin. Due to pressure the air bubbles travel upwards, and the monoterpenes get adsorbed on the resin surface due to affinity. Figure 5-2 Schematic of CESR setup Resins used for terpenoid capture Resins are polymeric compounds with tunable chemistry. The resin can be a homopolymer (made of a single type of repeating monomers) or heteropolymer (made of more than one type of repeating monomers). The physicochemical properties of the polymer depend on the monomeric units that it contains. Monomeric blends are chosen for desired characteristics like hydrophobicity/hydrophilicity, type of interaction, stability, porosity etc. Moreover, the resins can be tuned to be highly solvent resistant and can be regenerated for reuse. Adsorption is affinity based on weaker forces like van der Waals forces. It supports multilayer binding and is readily BioreactorAir flow outFBCDResinWaterSpargerAgitator135 reversible. Since, terpenoids are hydrophobic, adoption is preferred phenomenon for their recovery. Different resins were screened for their efficiency to capture carene. Resins employed in this study and their physicochemical properties are listed in Table 5.1. Purolite resins were gifts from Purolite, USA. Amberlite resins were purchased from Sigma. The resins were activated as per the supplier recommendations, washed thrice with water prior to use. Table 5.1 Resins employed in the study and their properties Resin Hydrophobicity Polymer structure Pore size (Å) Surface area (m2/g) Amberlite XAD-4 Hydrophobic Styrene-divinylbenzene 50 725 Amberlite XAD-7 Hydrophilic (moderately polar) Acrylic 90 450 Amberlite XAD-8 Hydrophilic (moderately polar) Acrylic ester 225 140 Purolite Purosorb PAD610 Hydrophilic (moderately polar) Polyacrylic-divinylbenzene 290 450 Purolite Macronet MN202 Hydrophobic Polystyrene-divinylbenzene 6-210 900 Purolite Macronet MN270 Hydrophobic Polystyrene-divinylbenzene 16-80 1100 136 Screening resin for carene capture 10 µL of carene was added to 1 mL of each resin dispersed in 9 mL water in an airtight glass vial and was shaken for 1 hour at 22 °C. The resin was then allowed to settle, and the solids were separated by filtration. Carene from the filtered water was extracted using dodecane extraction and analyzed as well. Hexane was used as a solvent to desorb carene from resin. The desorption was carried at room temperature. All the steps were done in sealed glass vials. 2 µL of hexane fractions were analyzed on GC-MS for characterization. The analytical method used is described in Section 4.2.4 (Page number 89). 137 5.3 Results and discussions Adsorption based carene recovery Desorption was carried out for 1 week. The results are reported in Table 5.2. Table 5.2 Screening resin for carene recovery Resin % Carene remaining in the water after extraction % Carene recovery (desorption time) 2 h 3 days 1 week Amberlite XAD-4 15.5 ± 1.5 58 ± 2 74 ± 4 72 ± 6 Amberlite XAD-7 4.1 ± 1.5 42 ± 4 71 ± 7 75 ± 11 Amberlite XAD-8 0.08 ± 2.5 35 ± 20 77 ± 9 69 ± 18 Purolite Purosorb PAD610 0.03 ± 0.0 84 ± 6 85 ± 13 93 ± 15 Purolite Macronet MN202 0.03 ± 0.0 57 ± 10 79 ± 7 86 ± 18 Purolite Macronet MN270 0.05 ± 0.5 58 ± 10 67 ± 13 66 ± 19 100 % carene was never recovered due to its volatility and loss by evaporation. Due to the same reason, carene desorption reduced on longer incubation for Amberlite XAD-4 and XAD-8. Purolite Purosorb PAD610 could recover 84 % of carene on 2 h of desorption and was selected for bioprocess optimization. PAD601 is made of a polyacrylic-divinylbenzene copolymer which is moderately hydrophilic. The analysis revealed that acrylate co-blocks aid desorption whereas, styrene co-blocks are important for adsorptive interactions. Since the resin can be reused, optimization of both adsorption efficiency and desorption time is necessary. PAD610 though could not be desorbed completely higher efficiency can be achieved when it is recycled. 138 Bioreactor parameter optimization The bioreactor parameters like air flow rate, agitation speed and resin bed volume were optimized. Optimal parameters are shown in Figure 5-3. The optimal conditions for monoterpene product capture were determined to be agitation speed of 250 rpm, air flow rate of 2 vvm and 7 mL resin. Higher rates of air flow through the resin bed were observed to dry up the resin. As a consequence, the FBCD was kept immersed in water. Figure 5-3 Bioreactor parameter optimization for efficient carene production and recovery 139 The bioreactor and FBCD set up is shown in Figure 5-4. Figure 5-4 Experimental apparatus Terpenoid product analyses The optimization was done for SACar strain. SAMyr strain was run on bioreactor as well on the optimized parameters. Table 5.3 summarizes the monoterpene product distributions obtained. The product distribution at bioreactor scale was compared with aerobic shake flask fermentation (reported in section 4.4.3). The results match closely but the bioreactor runs had higher variability. This suggests that the capturing mechanism is efficient for a variety of monoterpenes. This was also validated by an independent study of testing resin adsorption for a mixture of different monoterpenes. 140 Table 5.3 Product distribution comparison of shake flask and bioreactor scale fermentations Products SACar aerobic shake flask fermentations (Section 4.2.4) SACar bioreactor fermentations (This study) Carene 83.4±0.9 84.8±1.5 Terpinolene 3.9±0.2 3.9±0.6 Sabinene 8.5±0.2 7.4±0.9 Myrcene 2.5±0.2 2.2±0.7 Limonene 1.2±0.2 1.0±0.5 Terpinene 0.3±0.2 0.5±0.2 Pinene 0.1±0.1 0.1±0.1 SAMyr aerobic shake flask fermentations (Section 4.2.4) SAMyr bioreactor fermentations (This study) Myrcene 98.7±0.8 98.4±0.9 Carene 1.0±0.2 1.2±0.5 Pinene 0.3±0.1 0.4±0.6 Terpenoid recovery comparison The SACar fermentations in the bioreactor were run with 10 % (v/v) overlay with dodecane for 18 h after induction. 4.4 µg/L carene was recovered from the dodecane. When FBCD was employed for the capture replacing dodecane overlay, the carene recovery was 26.6 µg/L. A 6-fold improvement in the recovery was observed. Similarly, 14.6 µg/L myrcene was recovered from the 141 SAMyr fermentations. No monoterpenes were detected in media and water used to immerse FBCD. In conclusion, the terpenoids were successfully captured from the recombinant E. coli fermentation fluidized bed capturing device. The device was connected externally to the air flow outlet and was filled with resin. The desorption was carried out using hexane but other solvents like methanol can be used as well. Scale-up at this bioreactor scale improved the terpenoid production of SACar and SAMyr but the product distributions remained similar to that found in aerobic shake flask fermentations. 142 Chapter 6: Other scholarly contributions 6.1 A Biogenic Photovoltaic Material166 Other contributors: Sarvesh Kumar Srivastava, Przemyslaw Piwek, Arman Bonakdarpour, David P. Wilkinson, Vikramaditya G. Yadav The organic dye-sensitized solar cells (DSSCs)167 are fabricated by immobilizing Ru-based light-absorbing chelates over a layer of titania (TiO2) working electrode in presence of an electrolyte168. However, despite significant progress, they require toxic solvents, carcinogenic chemicals and expensive & rigid process controls under clean-room environment limiting their practical application. In fact, a combination of water-based electrolytes169 and naturally occurring dyes170 have been envisioned to hold the key for the development of solar cells and our future energy needs. The material that we have developed, in contrast, is biogenic171,172 and comprises of a porous mesh of E. coli BL21 cells that are encapsulated with TiO2 (Figure 6-1). The bacterial cells are genetically engineered to synthesize lycopene, a photosensitive dye, and TiO2 is deposited onto the cells via a tryptophan-mediated supramolecular interface to produce a core@shell-like morphology. Unlike other biological or bio-hybrid photovoltaic materials, not only is our synthetic scheme uncomplicated, but the resulting material is also exceptionally stable and exhibits impressive PV properties when used as an anode in an I-/I3--based DSSC. 143 Figure 6-1 Sequential representation of whole cell bio-PV materials. (a) molecular cloning of E. coli for expression of lycopene, (b) non-covalent surface binding of TiO2 nanoparticles resulting in core@shell-like morphology and (c) deployment of biogenic PV material towards DSSC fabrication. We recorded an open circuit (VOC) potential of 0.289 V, a short circuit (ISC) current of 0.19 mA and a corresponding short circuit current density (JSC) of 0.686 mA/cm2 (Figure 6-2(a)). Furthermore, since we coated TiO2 NPs around dye-encapsulating bacteria, we generate a preponderance of biogenic-hydrophobic interfaces that significantly increase the resistance. The time-dependent (or ON/OFF) illumination of the bio-PV DSSC (Figure 6-2(b)) exhibited a temporal variation of potential. When illuminated, a net potential difference of about ~ 0.2 V was observed over a period of 5 minutes with minimal decay. We also recorded cyclic voltammograms (CV) to confirm the PV effect (Figure 6-2(c)). When the illumination was turned off, we observed a typical capacitor-like charging-discharging behavior. However, when the DSSC is illuminated, we measure a current flow of ~ 5 µA (~ 0.02 mA/cm2). 144 Figure 6-2 Electrochemical measurements of bio-PV DSSC. (a) I-V curves, (b) open-circuit photovoltage response (time dependent) and (c) cyclic voltammetry curves The absence of any reduction-oxidation peaks suggests that the bio-PV material and associated DSSC are stable in the operational range of -0.1 to -0.2 V, which is also the corresponding range of the working potential. The use of metagenomics and synthetic biology to screen and clone microbial hosts that synthesize highly photoactive dyes, together with the flexibility and environmentally-friendly approach offered by biogenics (no further downstream processing, for instance), offers substantial advantages in the development of novel bio-PV materials and devices such as DSSCs, photodiodes and bio-polymeric cells. 145 6.2 Isolation of phenolic monomers from kraft lignin using magnetically recyclable TEMPO nanocatalyst173 Other contributors: Saurabh C Patankar, Li-Yang Liu, Lun Ji, Vikramaditya Yadav and Scott Renneckar A future bioeconomy will utilize platform chemicals sourced from biomass to create functional products and this action mirrors one central tenet of green chemistry. For example, vanillin has been suggested as a platform chemical that can be transformed into materials such as polyethylene terephthalate analogs useful in plastic containers to carpets and other textiles. While much research has been performed for second generation biorefineries producing cellulosic ethanol, a potential cornerstone of a bioeconomy, cellulosic ethanol targets have not been met to date. In part, biofuels have a poor valorization factor compared to platform chemicals such as vanillin and the environmental benefits have not been realized due to difficulties associated with scaling the technologies. Moreover, it is not clear there can be a return on investment of cellulosic ethanol biorefineries, prior to mass electrification of vehicle fleets occurring within the next decade. This issue ushers the existing pulp and paper industry to lead the crucial transformation towards the production of renewable feedstock for a global bioeconomy. However, mills have faced economic hurdles and many of them have closed in the last decade. In part, like second generation biorefineries, pulp and paper companies struggle to deal with a quarter to a third of the biomass which is composed of lignin. The majority of chemical pulps are produced from the kraft process, which uses sodium sulfate to delignify wood chips, breaking native C-O ether linkages in lignin and subsequently leaching its fragments into the alkaline pulping liquor. In this process, lignin gets modified with elemental sulphur and further gets repolymerized to develop into a 146 complicated macromolecule with considerable heterogeneity. This heterogeneity limits usage of lignin to its fuel value during chemical recovery and recycling of caustics. Moreover, this aromatic polymeric material has an important role in production of platform chemicals in the bioeconomy, yet there are limited economic compelling reasons to work with lignin. In North America, three different kraft lignins are available in semi-commercial quantities from three mills that all use gymnosperm species, which contain more than 90% guaiacyl lignin derived from the dehydrogenative polymerization of coniferyl alcohol. There are oxidative routes to isolate phenolic aldehydes from this material. However, the technologies employed require consumption of a large amount of alkali and high-temperature reactions with the pathway of vanillin cleavage. Deriving vanillin from biomass has low yields with large inputs of energy and material. Hence, novel catalytic oxidative depolymerization techniques have a crucial role in developing greener bio feedstock processes. In this work, we reported the use of novel catalyst designated as Fe@MagTEMPO developed by our collaborators for the oxidative depolymerization of lignin to obtain a higher yield of vanillin with lower calculated E factor than previously reported in the literature (Figure 6-3). Figure 6-3 Oxidative depolymerization of kraft lignin using the Fe@MagTEMPO catalyst 147 Table 6.1 Comparison of vanillin yield derived through oxidative depolymerization of kraft lignin using Fe@MagTEMPO catalyst with values in literature Lignin type Reaction Conditions Vanillin yield (%) E factor Ref. Kraft lignin WestVaco Co. (Pinus Spp.) 60 g.L-1 lignin, 2N NaOH aq. solution, 9 bar with pO2 being 3 bar, 130° C, 35 min 10.8 20.60 174 Lignosulfonate LS1200 110.6 g LS1200, NaOH solution (18 g NaOH in 16 cm3 water), Preheated at 15 bar O2, 190° C for 15 min 1.08 g CuSO4. 5H2O catalyst, 2 cm3 Nitrobenzene 12 bar O2, 190° C, Upto 30 min 5.9 19.14 175 Kraft lignin (Pinus spp.) LWest from WestVaco Co. 30 mg LWest, 7 cm3 of 2M NaOH aq. solution 0.45 cm3 Nitrobenzene, 170° C, 4 h 12.14 273.59 176 Sodium Lignosulfonate NaLS 220 g.L-1 NaLS, 3M NaOH aq. solution (pH 14) 4.6 g.L-1 copper sulfate (Cu2+), 11.5 bar with pO2 being 1.3 bar, Air flowrate of 4.5x10-3 m3.min-1 140-160° C 7 21.38 177 Spruce kraft lignin 140 mg lignin, 30 cm3 of 2 M NaOH aq. solution 10 mg LaMn0.8Cu0.2O3 catalyst, 5 bar O2 + 15 bar He 175° C, 10 min 17.3 217.73 178 Indulin AT from West Rock 1% (w/w) Lignin sample, 200 cm3 water as solvent 0.175 mg.cm-3 Fe@MagTEMPO recyclable catalyst 0.2 mmol NaBr, 5 mmol.g-1 NaClO, 25° C, 4 h 15 7.75 This work Acetone soluble Indulin AT (ASKL) 21 5.25 Acid wash Indulin AT 19.7 5.66 148 The Fe@MagTEMPO catalyst has free amine groups attached to magnetic nanoparticles that were observed to provide a local alkaline environment to abstract proton from phenolic hydroxyl in lignin, eliminating the use of alkali during oxidative depolymerisation. This aspect reduces the waste generation by over 98% compared to traditional methods of isolating vanillin from kraft liquor that were infamous for generating 160 kg alkali waste per kilogram of vanillin. 149 6.3 The V-factor: Towards a new metric for gauging the efficiency & profitability of manufacturing processes for the bioeconomy Other contributors: Saurabh C Patankar, Vikramaditya Yadav and Scott Renneckar Several metrics have been formulated to evaluate the environmental impact of chemical manufacturing processes. However, there are no formulas for quick, back-of-the-envelope estimation of their efficiency of resource utilization and profitability, which are hugely influential when it comes to determining investments. In this work, we developed a new metric called the V-factor to estimate these parameters for manufacturing processes that utilize biomass. Additionally, we also argued that co-assessment of E- and V-factors will greatly facilitate the development of environmentally and economically sustainable processes for the valorization of biomass (Figure 6-4). Figure 6-4 Comparison between E-factor and V-factor The unabridged V-factor incorporates all variables that influence the profitability of a manufacturing process, namely the costs of its reactants and products ($/kg), its fractional yield, 150 its capital and operating expenditures (CapEx and OpEx, respectively, $/kg for both), tonnage of the product (MT or metric tons), and the tonnage of its product sector (MT). The unabridged V-factor (Equation 6-1) is estimated as: Unabridged V-factor = Cost of productCost of reactant × Fractional yield × CapExOpEx × Tonnage of productTonnage of product sector Equation 6-1 The unabridged V-factor equation Although the unabridged V-factor adequately describes how efficiently a process valorizes its inputs, it is not easy to calculate. This is especially true for academic users who may not have good estimates for CapEx, or up-to-date information about pricing or product tonnages. It is also evident that the equation has redundancies that could be factored out in most cases. As a result, we have simplified the previous formula by incorporating the fractional dollar output for individual sectors. We term the simplified metric as the V-factor (Equation 6-2), and the fractional dollar output is an attribute of the industrial sector in which the product will be sold. V-factor = Cost of product Cost of reactant × Fractional yield × Fractional dollaroutput of sector Equation 6-2 V-factor equation This simplification greatly improves the utility of the V-factor and provides academic users with a useful tool to quickly assess profitability during the formulation of green chemistry and engineering research strategies. The fractional dollar output also ranges between 0 and 1. In addition, the sum of the fractional dollar outputs for all sectors of the chemical industry is one. 151 Forecasted global averages of fractional dollar outputs for some common industrial sectors between now and 2050 are listed in Table 6.2. The impacts of manufacturing volumes, capital and operating expenditures, and investments on R&D on the profits has been assessed previously 179,180, and it has been observed that a sector’s profit share of the global chemical market has remained constant over a period of 20 years and is expected to remain same over next 25 years. The volume or tonnage at which a product must be manufactured in order to attain economies of scale, the capital and operating expenditures, and investments on R&D that are typically required for a sector greatly influence its profit share. For instance, the profit share is positively correlated with manufacturing volumes. Sectors with higher manufacturing volumes such as petrochemicals typically achieve greater economies of scale compared with speciality chemical producers 181. However, higher manufacturing volumes usually incur substantially greater capital expenditures. As a consequence, the profit share for operating in a low-tonnage sector, despite having disadvantages such as higher product and process R&D expenditures, is not significantly lower in comparison. We used the sector’s profit share data and coined the term fractional dollar output to simplify the determination of V factor. The use of the fractional dollar output for estimating the profitability of processes is akin to performing a gate-to-gate life cycle analysis (LCA) in lieu of the more accurate cradle-to-grave LCA. 152 Table 6.2 Predicted global averages of fractional dollar outputs of selected sectors Sector Fractional dollar output Basic chemicals, fuels and petrochemicals 0.3 Pharmaceuticals and agrochemicals 0.2 Speciality chemicals 0.2 Polymers and fibres 0.2 Oleochemicals, surfactants & auxiliary chemicals 0.1 In this work, we demonstrated the use of the V factor in understanding the profitability of over 50 chemical manufacturing processes. 153 6.4 Microbial growth and its characterization on nanocellulose synthesized from cherry veneer Other contributors: Saurabh C Patankar, Muzaffer Karaaslan, Vikramaditya Yadav and Scott Renneckar The use of reusable TEMPO nanocatalyst (Fe@MagTEMPO) for the synthesis of nanofibrillated cellulose from kraft pulp182 and phenolic monomers from kraft lignins173 (Section 6.2) has been demonstrated. We wanted to investigate one pot fractionation and modification of biomass using the same catalyst. The veneer was an excellent model for wood chips owning to its nominal thickness and longitudinal arrangement of fibers. The reactions were carried out in 200 cm3 distilled water containing 2 g cherry veneer chips (50 x 10 x 0.5 mm) at pH 10. Sodium bromide (30 mg) and Fe@MagTEMPO catalyst (50 mg) were added to the dispersion and the dispersion was heated to desired temperature and oxidation was started by dropwise addition of sodium hypochlorite (10 cm3). The reaction was stopped when no further drop in pH was observed. The catalyst was separated from the reaction using an external magnet. It was ensured that the reaction mixture was still alkaline (pH 10) to keep the lignin fraction solubilized. The cellulose fraction was then separated across by filtration and redispersed in water such that solid concentration was 0.5 % (w/w). This dispersion was blended for 30 min and homogenized for two cycles to get nanocellulose. The nanocellulose samples were stored in a closed container at 4 °C. It appeared that the sample was infected with microbes after a duration of 10 months. This is the first report of characterization of contaminants of treated nanofibrillated cellulose. The nanocellulose sample was inoculated in tryptic soy broth (purchased from BD Biosciences) and modified Luria-Bertani broth (1% w/w tryptone, 1% yeast extract, 1% sodium 154 chloride). Cultures were grown for seven days at 22 °C, 30 °C and 37 °C. The cultures were streaked on tryptic soy agar media and modified Luria-Bertani agar media plates and incubated again for seven days at 22 °C, 30 °C and 37 °C. Morphologically distinguished colonies were isolated and analyzed using optical microscopy. Genomic DNA was extracted from the isolates by phenol/chloroform/isoamyl alcohol extraction. Cell pellets were washed and resuspended in 10mM Tris hydrochloride buffer (pH = 8) containing 1mM EDTA and 3 mg/mL lysozyme. The suspension was incubated at 60 °C for an hour. 15 ug/mL sodium dodecyl sulfate, Proteinase K and RNAase. Samples were incubated at 60 °C for an hour. Phenol solution (equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA) was added to samples. All samples were centrifuged at 12000 rpm to collect aqueous layer containing DNA. The layer was washed with a mixture of chloroform and isoamyl alcohol several times to remove phenol. The purity of the DNA was assessed by the ratios of absorbance at 260 nm/280 nm and 260 nm/230 nm on Thermo Fisher Scientific NanoDrop™ 2000/c Spectrophotometer. Genomic DNA was stored at -20 °C until further steps. Ribosomal RNA (rRNA) gene sequencing technique was used for phylogenetic characterization of the four isolated colonies. Three sets of primers were used to amplify rRNA gene. 16S_F (5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACG GGNGGCWGCAG -3’) and 16S_R(5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGA CAGGACTACHVGGGTATCT-AATCC-3’) primers (Tm of 60 °C) target bacterial 16S V3 and V4 region183 amplifying region of 500-600 bp.ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) primers (Tm of 57 °C) target highly variable fungal Internal Transcribe Sequences (ITS) surrounding the 5.8S-coding sequence that is situated 155 between the Nuclear Small rDNA and Nuclear Large rDNA of the ribosomal operon184 amplifying region of 800-1000 bp. (GS primer info). Amplification was performed on extracted genomic DNA as a template by Phusion High-Fidelity DNA Polymerase in Phusion HF Buffer from Thermo Fisher Scientific. PCR conditions were as follows: initial denaturation at 98 °C for 3 min; 30 cycles of 10 sec denaturation at 98 °C, 30 sec primer annealing at a primer-specific melting temperature (Tm), 30 sec extension at 72 °C; final annealing for 10 min. The amplified DNA was separated on agarose gel by electrophoresis and extracted using the GeneJET Gel Extraction Kit by Thermo Fisher Scientific. The purified amplicons were sequenced from Genewiz Inc. The sequences obtained were aligned to NCBI gene bank database using the Basic Local Alignment Search Tool (BLAST). The sequence alignments that elicited 100% query coverage with 100% identity and 0.0 E value were analyzed further. rDNA sequencing analysis characterized the four isolated contaminants as one bacterium and three fungi. Paraburkholderia fungorum strain OX1216 was identified betaproteobacteria. Whereas, Papiliotrema flavescens strain 7 (also known as Cryptococcus flavescens) and Rhodotorula mucilaginosa strain F.P705162591 were identified as yeast belonging to basidiomycota and basidiomycete classes respectively. The fourth multicellular filamentous fungus was identified as Penicillium chrysogenum strain HGQ6. Paraburkholderia fungorum and Papiliotrema flavescens were isolated as pure cultures. Rhodotorula mucilaginosa and Penicillium chrysogenum were not possible to isolate from their co-cultures with Paraburkholderia fungorum. Paraburkholderia sp. is reported to occur in symbiotic relationship with yeast and fungus185. Various strains of Papiliotrema flavescens possess the glycoside hydrolase activity186. Bacterial strain Paraburkholderia fungorum is reported to exhibit β-galactosidase activity187. 156 Atomic force microscopy (AFM) images were captured using Veeco multimode 8 system and RTESPA-525 cantilever tip with force constant of 200 N.m-1 and resonant frequency of 525 kHz. A drop of 0.001% (w/w) suspension of ligno-nanocellulose in water was put on freshly cleaved mica sheet and dried in air at 20 °C before AFM analysis. The AFM images were captured just after the synthesis of nanocellulose and after 10 months of storage in closed container at 4 °C. Figure 6-5 shows the AFM height image of freshly prepared nanocellulose synthesized from cherry veneer using Fe@MagTEMPO catalyst. It is visible that cellulose nanofibrils are individually separated and well dispersed on the mica due to the negative surface charge introduced after oxidation. The width of nanofibrils estimated from the height images are in the range of 2-5 nm, which is in accordance with the previous reports188. Figure 6-5 shows the height image of nanocellulose sample after 10 months of storage at 4 °C. It can be clearly seen that the nanofibrils were broken down to smaller pieces and shorter fibrils and chunks of these nanofibrils were aggregated. Figure 6-5 AFM height profile of nanocellulose samples synthesized from cherry veneer using Fe@MagTEMPO catalyst. (a) Freshly synthesized nanocellulose sample and (b) nanocellulose sample stored at 4 °C for 10 months 157 In addition, the assembly of microorganisms on the cellulose nanofibrils was identified by AFM (Figure 6-6). The AFM height as well as adhesion and peak force error images showed that the microbial growth and assembly of microorganisms specifically on individual nanofibrils. The dimensions of the particles on nanocellulose samples were approximately 200-500 nm in width and 20-80 nm in height. Figure 6-6 AFM image showing assembly of microbes on nanocellulose fibrils synthesized from cherry veneer using Fe@MagTEMPO catalyst 158 Chapter 7: Conclusions Terpenoids are a diverse class of natural products with great industrial potential but are produced in their natural hosts such as plants in low quantities. These natural sources fail to meet the increasing demand for these molecules. Total chemical syntheses of terpenoids have been attempted but usually involves low yielding steps requiring hazardous chemicals. The natural biochemistry is still the best path in terms of modularity and ability for widespread adoption for terpenoid production. Application of microbial cell factories for terpenoid biosynthesis has provided an alternative with advancements in the synthetic biology and metabolic engineering tools. To design the system efficiently, a thorough knowledge of the biosynthesis is necessary. The terpenoid biosynthesis route in the natural hosts contains an upstream pathway of synthesis of C5 precursors IPP and DMAPP from central carbon metabolism; and a downstream pathway of polymerization of the precursors followed by cyclization and activation. There are two upstream pathways for terpenoid production- MVA pathway and MEP pathway. The MEP pathway is energetically balanced and more efficient than the MVA pathway in the utilization of sugars28. MEP pathway is also natively expressed in bacteria which have proven to be excellent heterologous hosts for terpenoid biosynthesis25,79,118. The yields of terpenoids through such platforms are still lower than their theoretical maxima and various strategies have been undertaken to optimize and improve the flux through the pathway. These efforts for improving the heterologous terpenoid production will be in vain if the large-scale bioprocess development is not possible. Lack of suitable terpenoid recovery technique is impeding the terpenoid commercialization. Hence, I set the objective to 159 design and improve biocatalysis and bioprocess for terpenoid production in E. coli in a holistic way. The overall question was ‘despite tremendous economic advantages, why there are very few examples of commercial terpenoid production?’ This question was broken down into three well-identified research objectives. The first objective sought to augment the overall terpenoid production through improvement in the intracellular pool of C5 precursors – IPP and DMAPP. The second objective was to study the terpene synthase catalysis with a focus on the factors governing their promiscuity, and the third objective was to design an efficient terpene recovery technique for large scale bioprocessing. 7.1 Improvement in the intracellular pool of C5 precursors The MEP pathway is the first bottleneck in the biosynthetic route. Various strategies have been undertaken to optimize and improve the MEP pathway flux. The reconstructed the MEP pathway operon from native MEP pathway genes achieved similar performance to previously reported best-in-class constructs that used multivariate modular metabolic engineering for taxol precursor biosynthesis28. This construct was considered a basis in the study of augmenting C5 precursor availability. Isoprene and lycopene were used as the reporter molecules. The strategy used for enhancement of the reporter terpenoids through MEP pathway engineering involved a metagenomic search for more active and stable orthologs of pathway bottleneck enzymes – Dxs, IspD, IspF and Idi. The recent discoveries of IspDF fusion enzymes have predicted to channel the substrate from one active site to the next and decrease diffusional limitations. The metagenomic search hence conducted was tailored towards the search for such enzyme fusions. Three fusions were discovered and all co-localized IspD and IspF active sites onto 160 a single scaffold. When these fusions were tested, one fusion (IspDF1) increased the lycopene titers by 17 % and isoprene titers by 73 %. When the cell densities were factored in, IspDF1 construct enhanced the normalized titers by 9 % and 60 % for lycopene and isoprene respectively. The fusion not only reduced the metabolic burden but also enhanced terpenoid production. But this differential behavior for hemiterpenes like isoprene and tetraterpene like lycopene remained unexplained. The improvement in the flux on fusion expression was initially contributed to the co-localization effect that channeled the substrate effectively through the pathway. This directed the next study on the role linkers play in the effectiveness of the enzyme fusion. This study was conducted on non-natural enzyme chimeras of IspDF1 as well as E. coli IspD and IspF. Four different linkers (flexible linker, rigid linker, linker from cjIspDF and linker from IspDF1) were tested. The rigid linker for IspDF1 chimera improved the normalized lycopene titers by 34 % but lowered overall production by 21 %. This was due to the lower cell growth rate that reflected in lower OD600 of the cultures. Whereas for non-natural fusions of native IspD and IspF fusion chimeras, none of the fusions showed a positive effect. In the study so far, the role of IspE which catalyzes the intermediary step was unclear. Hence, a similar set of experiments were done for the operon now also overexpressing IspE. To our surprise, this had a net negative effect on lycopene production. This observation was attributed to metabolic stress. Higher normalized titers for fusion of IspD1 and IspF1 linked by the rigid linker directed us to separate the fusion as two genes each expressing the domain individually. This exhibited the outcomes similar to that of the fused enzyme. When IspE was overexpressed along with the operon comprising the best performing fusion, normalized titers increased by 23 % but the overall lycopene titers dropped by 31 %. This concluded that the individual domains in the IspDF1 domain were higher active and the effect of fusion was no more than reducing transcriptional burden. 161 Another approach of linking enzymes catalyzing consecutive steps in the MEP pathway was then considered to augment C5 precursor pool in E. coli. The IspDE and IspEF fusions with flexible linker were constructed for native E. coli enzymes. The IspDFLEF fusion produced lycopene at the highest titers of 281 mg/L. This titer is 20% higher than the basal strain SALyc-SDFI (overexpressing Dxs, IspD, IspF and Idi), is 2.3-fold strain SALyc-SDFEI (overexpressing Dxs, IspD, IspF, IspE and Idi) and is 70-fold SALyc strain (that utilizes the genomic MEP pathway and does not contain any additional MEP gene cassette). In this work, we demonstrated the strategy of non-naturally co-localizing enzymes involved to enhance the flux through the pathway thereby increasing the intracellular precursor pool. 7.2 Understanding the biocatalytic sequence of steps in the MEP pathway The IspDF1 fusion improved the flux through the MEP pathway but IspDF2 and IspDF3 lowered it. Moreover, IspDFLE fusion was highly effective that its unfused variant. These results put forth the question on the relevance of the natural existence of fusions of IspDF that catalyze non-consecutive steps in the pathway. Here I proposed a bifurcation in the step wherein, IspE can also precede IspD in the catalytic cascade. The in vitro reactions of IspE were analyzed by ATP bioluminescence assay, HPLC and LC-MS. The analysis led to the characterization of ME-2,4-PP as a product. The chemical synthesis of ME-2,4-PP was challenging and failed, and hence the final product could not be characterized in greater detail. In addition to this, the IspE reactions accepted both ATP and ADP as phosphate donor. This study opened a possibility for potential plasticity in the MEP pathway. 162 The strategy employed to improve the flux through the MEP pathway is not limited to only the MEP pathway but has wider applicability. Moreover, this study also guided the further investigation of the pathway cascade. Though much more work needs to be done to verify it in detail, this is the first report of the existence of the MEP pathway bifurcation. The robust and high precursor producing strain can not only be employed to improve terpenoid yield but also used to mine newer terpenoids that otherwise remain undetected in the natural host due to lower abundance. 7.3 Study of terpene synthase biocatalysis Terpene synthases are reported to possess catalytic promiscuity that converts single substrate to an array of products that are usually structural isomers of each other. The strategies of improvement of precursor supply are in vain if the TS enzymes downstream are not selectively utilizing the precursors to make only the desired product. Few examples are TS are highly selective but there is not a selective enzyme for all the terpenoids known. Hence, the only option available is to engineer TSs to reduce their promiscuity. Individual attempts have been made in this direction and have proven to be useful137,156,189,190. The fact often ignored is the natural hosts such as plants that are the source of these enzymes display the presence of more than one TSs. Evolution has diversified the enzymes to achieve newer functions. The objective of this study was to investigate TS promiscuity in the framework of C5 precursor abundance. The factor of evolutionary pressure was kept constant by selecting four enzymes carene synthase, myrcene synthase, limonene synthase and linalool synthase from Norway spruce. We reported the successful expression of MyrTS, LimTS and LinTS in E. coli and characterization of the product spectra. 163 The first conclusion of this work was that the product distribution changed when studied in vivo. In addition, MyTS which was reported to be highly selective enzyme136 in vitro reactions with GPP, made carene and pinene as by-products in vivo. Newer products were characterized for other TSs as well. The study also exhibited the influence of fermentation parameters like oxygen supply on product distribution. The highest terpenoid titer achieved for CarTS was 12.64 µg/L carene, for MyrTS was 0.84 µg/L myrcene, for LimTS was 1.08 µg/L limonene and for LinTS was 10.33 µg/L linalool. The substrate and TSS docking studies on homology models generated for the enzymes revealed the plausible role of amino acid residues taking part in the active site structure. The residues like arginine were dominant in CarTS and LimTS that form cyclic products; whereas, residues like phenylalanine were dominant in MyrTS and LinTS that form acyclic products. The conclusions from this comprehensive study can be utilized to engineer each one of them for the desired activity. 7.4 Bioprocess development for terpenoid recovery There are many lab scale successes of efficient strain engineering for terpenoid production. But the scale up is challenging mainly due to the product recovery technique (such as biphasic extraction) that is suitable at a small scale but loses the process profitability at a large scale. The module – fluidized bed capture device (FBCD) was designed that required minimal hardware perturbations for integration with the bioreactor. The device design was made to utilize volatility and hydrophobicity of the terpenoids to recover them from the fermentation. The device contains resin bed fluidized in water by the air flow out from the bioreactor. The terpenoids in the air outlet were efficiently captured by adsorption. The terpenoids were then recovered from the resin by desorption using solvents like methanol and hexane. The terpenoid products then can be distilled 164 and the solvents can be recycled. Methanol and hexane are defined in class II of restricted solvents in pharmaceuticals by ICH (International Council for Harmonisation) Q3C guidelines, and their residual content should be closely monitored in the final product. The process can also be tailored to utilize class III solvents like acetone, isopropanol, pentane which are Generally Recognized As Safe (GRAS). The recovery of carene increased six-fold when the dodecane based biphasic in situ extraction was replaced by FBCD. The process was validated for SACar and SAMyr strains; and their product distributions were similar to that obtained in the aerobic shake flask fermentations in my previous study. The titers of major terpenoid achieved with the optimized bioprocess with FBCD were 26.6 µg/L for SACar and 14.6 µg/L for SAMyr. The fold improvement was a result of improved aeration in the system as well as reduced product toxicity. This architecture developed and studied for terpenoid bioprocesses, finds wider applications for products with similar characteristics like butanol and alkanes. In conclusion, the entire work was conducted in a holistic way to address the existing challenges in the terpenoid biomanufacturing. Various expertise gained during this work were utilized to study problems in the competing fields that are summarized in Chapter 6 of the thesis. 165 7.5 Future work This work was based on addressing three major challenges in the field of study. The work led to many novel outcomes and observations. Further experiments and tests towards bolstering claims in this thesis as well as providing depth to the scientific findings can be carried out. This chapter discusses different aspects of these future efforts in detail. Improvement in MEP pathway flux (Chapter 2) Instead of using reporter terpenoids and indirect measurements of the pathway flux, a more insightful study could be carried using radio- or isotopically labelled substrates. The in vivo kinetic parameter estimation for each catalytic step would provide greater insights into the flux determination. Another simpler approach would be to use defined carbon source (like glucose or glycerol) in minimal media and quantify yield for the reporter terpenoids. The present work did not consider this as an option due to slower growth rates in minimal media. The metagenomic bifunctional enzyme screening could be applied to different genomic libraries to identify suitable targets. The advanced tools can be used to effectively develop homology models for the natural as well as non-natural enzymes. The best performing fusions IspDF1 and IspDFLE could be purified to obtain crystal structures and to study the effect of substrate channeling further. To evaluate the observations of IspE overexpression, the transcriptomic analysis would reveal its influence on cellular activity as well as the fair comparison can then be carried out on normalizing the terpenoid titers with individual enzyme expression levels. Moreover, parallel experiments could be carried out in cell-free systems and the optimal MEP pathway enzyme concentrations could be quantified. 166 The activity of IspD1 and IspF1 domains can be estimated and depending on the results, more study can be conducted by replacing each of the domain with E. coli monofunctional enzyme for example fusions of IspD1 and E. coli IspF. Such a study can be extensively carried out with domains of other natural fusions as well as non-natural IspE fusions. As a step forward, the sequence-activity correlations would result in evolving the IspD and IspF enzymes for higher activity. These experiments in totality can also guide the engineering of MEP pathway for improved terpenoid production. MEP pathway biochemistry (Chapter 3) The study undertaken in this work was rather serendipitous and for proof-of-concept. The detailed analysis of the MEP pathway cascade is necessary in order to confirm. The direction of the future work would be towards detail analysis of each reaction step using a isotopically labeled substrate. There are no reports of testing MEP as a substrate for IspE. In vivo product characterization of these steps can add great value to the work in the direction of understanding the MEP pathway. ME-2,4-PP chemical synthesis can be pursued further with the newer approach and the products can be characterized in detail. Terpene synthase study (Chapter 4) The bioprocessing parameters like oxygenation and pH that influenced the titer of terpenoid, and the product spectra could be studied for other TSs to deduce the exact mechanism. The MTSs studied could be purified and crystal structure could be determined to reveal the active site architecture. The observations of the homology modeling and structure-function correlations can also be verified further with mutations. The product spectra of the mutants can be correlated 167 back to the structure of the active site. The comprehensive study would then generate an excellent model to engineer monoterpene synthases. The strategy of comparison of in silico observation with in vivo product profile of the MTSs can be applied to other species of plants as well to other classes of TSs. A database of such observations can be used to direct the evolution studies for improvement of TS activity. Terpenoid recovery from the bioprocess (Chapter 5) The efficiency of the developed capture technique could be improved further in several ways. The fluidized bed height can be increased to allow longer contact of the rising air bubbles with water as well as resin beads. A gas distributor plate can be added at each of the nozzle and an air bubble breaker can be used that will distribute the bubbles in smaller sizes increasing the area for stripping. Employment of ways by which the beads will be in direct contact with the culture could highly improve the recovery. I attempted this by dispersing beads in the fermentation media but the growing cells clogged the pores moreover, downstream processing and recovery of terpenoids were cumbersome. Another approach will be- use porous adsorbent pads either integrated into the bioreactor headspace or covering the top layer of culture. For later to work, the material of adsorbent has to be resistant to biofilm formation. More work on this topic is currently being pursued by our collaborators. 168 Bibliography 1. The Global Chemical Industry: Catalyzing Growth and Addressing Our World’s Sustainability Challenges. (2019). 2. Christianson, D. W. Structural and Chemical Biology of Terpenoid Cyclases. Chemical Reviews 117, 11570–11648 (2017). 3. Christmann, M. Otto Wallach: Founder of Terpene Chemistry and Nobel Laureate 1910. Angew. Chemie Int. Ed. 49, 9580–9586 (2010). 4. KEGG PATHWAY: Terpenoid backbone biosynthesis - Reference pathway. Available at: https://www.genome.jp/kegg-bin/show_pathway?map00900. (Accessed: 8th February 2019) 5. Leavell, M. D., McPhee, D. J. & Paddon, C. J. Developing fermentative terpenoid production for commercial usage. Current Opinion in Biotechnology 37, 114–119 (2016). 6. Hirai, S., Utsugi, M., Iwamoto, M. & Nakada, M. Formal total synthesis of (-)-taxol through Pd-catalyzed eight-membered carbocyclic ring formation. Chem. - A Eur. J. 21, 355–359 (2015). 7. Moritz, B. J., Mack, D. J., Tong, L. & Thomson, R. J. Total synthesis of the isodon diterpene sculponeatin N. Angew. Chemie - Int. Ed. 53, 2988–2991 (2014). 8. Long, R. et al. Asymmetric total synthesis of (-)-lingzhiol via a Rh-catalysed [3+2] cycloaddition. Nat. Commun. 5, 5707 (2014). 9. Justicia, J. et al. Bioinspired terpene synthesis: A radical approach. Chemical Society Reviews 40, 3525–3537 (2011). 10. Maimone, T. J. & Baran, P. S. Modern synthetic efforts toward biologically active terpenes. Nature Chemical Biology 3, 396–407 (2007). 11. Yoder, R. A. & Johnston, J. N. A case study in biomimetic total synthesis: Polyolefin carbocyclizations to terpenes and steroids. Chemical Reviews 105, 4730–4756 (2005). 12. Lévesque, F. & Seeberger, P. H. Continuous-flow synthesis of the anti-malaria drug artemisinin. Angew. Chemie - Int. Ed. 51, 1706–1709 (2012). 13. Walji, A. & MacMillan, D. Strategies to Bypass the Taxol Problem. Enantioselective Cascade Catalysis, a New Approach for the Efficient Construction of Molecular Complexity. Synlett 2007, 1477–1489 (2007). 14. Purkayastha, S., Markosyan, A., … J. M.-U. P. A. 15 & 2017, U. Stevia composition to improve sweetness and flavor profile. Google Patents (2016). 15. Palmer, P. I. et al. Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature). Atmos. Chem. Phys. Discuss. 6, 107–173 (2010). 16. Schindler, J. Terpenoids by Microbial Fermentation. Ind. Eng. Chem. Prod. Res. Dev. 21, 537–539 (1982). 17. Schmidt-Dannert, C. Biosynthesis of terpenoid natural products in fungi. Adv. Biochem. Eng. Biotechnol. 148, 19–61 (2015). 18. Reiling, K. K. et al. Mono and diterpene production in Escherichia coli. Biotechnol. Bioeng. 87, 200–212 (2004). 19. Martin, V. J. J., Pitera, D. J., Withers, S. T., Newman, J. D. & Keasling, J. D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003). 169 20. Eisenreich, W., Bacher, A., Arigoni, D. & Rohdich, F. Biosynthesis of isoprenoids via the non-mevalonate pathway. Cellular and Molecular Life Sciences 61, 1401–1426 (2004). 21. Kim, E. M., Eom, J. H., Um, Y., Kim, Y. & Woo, H. M. Microbial synthesis of myrcene by metabolically engineered Escherichia coli. J. Agric. Food Chem. 63, 4606–4612 (2015). 22. Tholl, D. Biosynthesis and biological functions of terpenoids in plants. Adv. Biochem. Eng. Biotechnol. 148, 63–106 (2015). 23. McGarvey, D. J. & Croteau, R. Terpenoid metabolism. Plant Cell 7, 1015–1026 (1995). 24. Vranová, E., Coman, D. & Gruissem, W. Network Analysis of the MVA and MEP Pathways for Isoprenoid Synthesis. Annu. Rev. Plant Biol. 64, 665–700 (2013). 25. Frank, A. & Groll, M. The Methylerythritol Phosphate Pathway to Isoprenoids. Chem. Rev. 117, 5675–5703 (2017). 26. Morrone, D. et al. Increasing diterpene yield with a modular metabolic engineering system in E. coli: Comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85, 1893–1906 (2010). 27. Yadav, V. G., De Mey, M., Giaw Lim, C., Kumaran Ajikumar, P. & Stephanopoulos, G. The future of metabolic engineering and synthetic biology: Towards a systematic practice. Metab. Eng. 14, 233–241 (2012). 28. Ajikumar, P. K. et al. Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli. Science (80). 330, 70–74 (2010). 29. Yokota, A. & Sasajima, K. I. Formation of l-deoxy-D-threo-pentulose and 1-deoxy-L-threo- pentulose by cell-free extracts of microorganisms. Agric. Biol. Chem. 48, 149–158 (1984). 30. Yokota, A. & Sasajima, K. I. Formation of 1-deoxy-ketoses by pyruvate dehydrogenase and acetoin dehydrogenase. Agric. Biol. Chem. 50, 2517–2524 (1986). 31. Rohmer, M. The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep. 16, 565–574 (1999). 32. Zhou, D. & White, R. H. Early steps of isoprenoid biosynthesis in Escherichia coli. Biochem. J. 273, 627–634 (1991). 33. Sprenger, G. A. et al. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc. Natl. Acad. Sci. 94, 12857–12862 (1997). 34. Lois, L. M. et al. Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 95, 2105–10 (1998). 35. Ye, L., Lv, X. & Yu, H. Engineering microbes for isoprene production. Metabolic Engineering 38, 125–138 (2016). 36. Banerjee, A. et al. Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway. J. Biol. Chem. 288, 16926–16936 (2013). 37. Ghirardo, A. et al. Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene. PLANT Physiol. 165, 37–51 (2014). 38. Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 295, 517–524 (1993). 39. Rohmer, M., Seemann, M., Horbach, S., Bringer-Meyer, S. & Sahm, H. Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate 170 pathway for terpenoid biosynthesis. J. Am. Chem. Soc. 118, 2564–2566 (1996). 40. Rohdich, F. et al. Cytidine 5’-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol. Proc. Natl. Acad. Sci. 96, 11758–11763 (1999). 41. Lüttgen, H. et al. Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol. Proc. Natl. Acad. Sci. U. S. A. 97, 1062–7 (2000). 42. Lange, B. M. & Croteau, R. Isopentenyl diphosphate biosynthesis via a mevalonate-independent pathway: isopentenyl monophosphate kinase catalyzes the terminal enzymatic step.file:///C:/Users/kwgeorge/Downloads/1-s2.0-S0006291X06000167-main.pdf. Proc. Natl. Acad. Sci. U. S. A. 96, 13714–9 (1999). 43. Herz, S. et al. Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Proc. Natl. Acad. Sci. U. S. A. 97, 2486–2490 (2000). 44. Richard, S. B. et al. Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway. J. Biol. Chem. 277, 8667–72 (2002). 45. Kipchirchir Bitok, J. & Meyers, C. F. 2 C-methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity. ACS Chem. Biol. 7, 1702–1710 (2012). 46. Hecht, S. et al. Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein. Proc. Natl. Acad. Sci. 98, 14837–14842 (2001). 47. Seemann, M. et al. Isoprenoid biosynthesis through the methylerythritol phosphate pathway: The (E)4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) is a [4Fe-4S] protein. Angew. Chemie - Int. Ed. 41, 4337–4339 (2002). 48. Lange, H., Kaut, A., Kispal, G. & Lill, R. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. Proc. Natl. Acad. Sci. U. S. A. 97, 1050–1055 (2000). 49. Altincicek, B. et al. LytB, a novel gene of the 2- C -methyl- D -erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli. FEBS Lett. 499, 37–40 (2001). 50. Rohdich, F. et al. The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. 100, 1586–1591 (2003). 51. Heider, S. A. E., Wolf, N., Hofemeier, A., Peters-Wendisch, P. & Wendisch, V. F. Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum. Front. Bioeng. Biotechnol. 2, 28 (2014). 52. Shin, B.-K., Ahn, J.-H. & Han, J. N -Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity. Bioinorg. Chem. Appl. 2015, 1–8 (2015). 53. Kwon, M., Shin, B. K., Lee, J., Han, J. & Kim, S. U. Characterization of Burkholderia glumae BGR1 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), the terminal enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. J. Korean Soc. Appl. Biol. Chem. 56, 35–40 (2013). 54. Puan, K. J., Wang, H., Dairi, T., Kuzuyama, T. & Morita, C. T. fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. 171 FEBS Lett. 579, 3802–3806 (2005). 55. Hahn, F. M., Baker, J. A. & Poulter, C. D. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase. J. Bacteriol. 178, 619–24 (1996). 56. de Ruyck, J., Durisotti, V., Oudjama, Y. & Wouters, J. Structural role for Tyr-104 in Escherichia coli isopentenyl-diphosphate isomerase: site-directed mutagenesis, enzymology, and protein crystallography. J. Biol. Chem. 281, 17864–9 (2006). 57. Wouters, J. et al. Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors. J. Biol. Chem. 278, 11903–11908 (2003). 58. Brammer, L. A., Smith, J. M., Wades, H. & Meyers, C. F. 1-Deoxy-D-xylulose 5-phosphate synthase catalyzes a novel random sequential mechanism. J. Biol. Chem. 286, 36522–36531 (2011). 59. Fox, D. T. & Poulter, C. D. Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli. Biochemistry 44, 8360–8368 (2005). 60. Rohdich, F. et al. Biosynthesis of terpenoids: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase from tomato. Proc. Natl. Acad. Sci. 97, 8251–8256 (2000). 61. Tang, M., Odejinmi, S. I., Allette, Y. M., Vankayalapati, H. & Lai, K. Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2- C-methyl-d-erythritol (CDP-ME) kinase of Gram-negative bacteria. Bioorganic Med. Chem. 19, 5886–5895 (2011). 62. Geist, J. G. et al. Thiazolopyrimidine inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum. ChemMedChem 5, 1092–1101 (2010). 63. Xiao, Y., Zahariou, G., Sanakis, Y. & Liu, P. IspG Enzyme activity in the deoxyxylulose phosphate pathway: Roles of the iron-sulfur cluster. Biochemistry 48, 10483–10485 (2009). 64. Xiao, Y., Chu, L., Sanakis, Y. & Liu, P. Revisiting the IspH Catalytic System in the Deoxyxylulose Phosphate Pathway: Achieving High Activity. J. Am. Chem. Soc. 131, 9931–9933 (2009). 65. Bian, G., Ma, T. & Liu, T. In Vivo Platforms for Terpenoid Overproduction and the Generation of Chemical Diversity. in Methods in Enzymology 608, 97–129 (Academic Press, 2018). 66. Gabrielsen, M. et al. Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis. J. Biol. Chem. 279, 52753–61 (2004). 67. Testa, C. A. & Brown, M. J. The methylerythritol phosphate pathway and its significance as a novel drug target. Curr. Pharm. Biotechnol. 4, 248–59 (2003). 68. Testa, C. A., Cornish, R. M. & Poulter, C. D. The Sorbitol Phosphotransferase System Is Responsible for Transport of 2-C-Methyl-D-Erythritol into Salmonella enterica Serovar Typhimurium. J. Bacteriol. 186, 473–480 (2004). 69. Puan, K. J. et al. Preferential recognition of a microbial metabolite by human Vγ2Vδ2 T cells. Int. Immunol. 19, 657–673 (2007). 70. Connolly, D. M. & Winkler, M. E. Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(delta 2-isopentenyl)-adenosine tRNA modification, and 172 spontaneous mutagenesis in Escherichia coli K-12. J. Bacteriol. 171, 3233–46 (1989). 71. Okada, K. et al. The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli. J. Bacteriol. 179, 3058–3060 (1997). 72. Apel, A. R., Ouellet, M., Szmidt-Middleton, H., Keasling, J. D. & Mukhopadhyay, A. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae. Sci. Rep. 6, (2016). 73. Jo, S. et al. Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production. J. Biotechnol. 258, 69–78 (2017). 74. Lim, J. H. & Jung, G. Y. A simple method to control glycolytic flux for the design of an optimal cell factory. Biotechnol. Biofuels 10, 160 (2017). 75. Bakker, B. M., Westerhoff, H. V, Opperdoes, F. R. & Michels, P. A. . Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs. Molecular and Biochemical Parasitology 106, 1–10 (2000). 76. Alberty, R. A. Systems of biochemical reactions from the point of view of a semigrand partition function. Biophys. Chem. 93, 1–10 (2001). 77. Liu, H. et al. MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli. Microb. Cell Fact. 13, 135 (2014). 78. Farmer, W. R. & Liao, J. C. Precursor balancing for metabolic engineering of lycopene production in escherichia coli. Biotechnol. Prog. 17, 57–61 (2001). 79. Xue, D., Abdallah, I. I., de Haan, I. E. M., Sibbald, M. J. J. B. & Quax, W. J. Enhanced C30carotenoid production in Bacillus subtilis by systematic overexpression of MEP pathway genes. Appl. Microbiol. Biotechnol. 99, 5907–5915 (2015). 80. Munoz-Bertomeu, J., Arrillaga, I., Ros, R. & Segura, J. Up-Regulation of 1-Deoxy-D-Xylulose-5-Phosphate Synthase Enhances Production of Essential Oils in Transgenic Spike Lavender. PLANT Physiol. 142, 890–900 (2006). 81. Lv, X. et al. Combinatorial pathway optimization in Escherichia coli by directed co-evolution of rate-limiting enzymes and modular pathway engineering. Biotechnol. Bioeng. 113, 2661–2669 (2016). 82. Zhou, K., Zou, R., Stephanopoulos, G. & Too, H.-P. Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production. Microb. Cell Fact. 11, 148 (2012). 83. Gabrielsen, M. et al. Biosynthesis of isoprenoids a bifunctional IspDF enzyme from Campylobacter jejuni. Eur. J. Biochem. 271, 3028–3035 (2004). 84. Testa, C. A., Lherbet, C., Pojer, F., Noel, J. P. & Poulter, C. D. Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway. Biochim. Biophys. Acta - Proteins Proteomics 1764, 85–96 (2006). 85. Lherbet, C., Pojer, F., Richard, S. B., Noel, J. P. & Poulter, C. D. Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway. Biochemistry 45, 3548–3553 (2006). 86. Brown, E. D., Walsh, C. T., Mindiola, D. J., Gehring, A. M. & Lees, W. J. Acetyltransfer Precedes Uridylyltransfer in the Formation of UDP- N -acetylglucosamine in Separable Active Sites of the Bifunctional GlmU Protein of Escherichia coli †. Biochemistry 35, 579–585 (2002). 87. Wilding, E. I. et al. Identification, evolution, and essentiality of the mevalonate pathway for 173 isopentenyl diphosphate biosynthesis in gram-positive cocci. J. Bacteriol. 182, 4319–4327 (2000). 88. Hedl, M. et al. Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis. J. Bacteriol. 184, 2116–2122 (2002). 89. Velayos, A., Eslava, A. P. & Iturriaga, E. A. A bifunctional enzyme with lycopene cydase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides. Eur. J. Biochem. 267, 5509–5519 (2000). 90. Arrach, N., Fernandez-Martin, R., Cerda-Olmedo, E. & Avalos, J. A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces. Proc. Natl. Acad. Sci. 98, 1687–1692 (2001). 91. CLARKE, J. H. et al. Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates. Biochem. J. 319, 515–520 (2015). 92. Huang, Z., Li, G., Zhang, C. & Xing, X. H. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library. Enzyme Microb. Technol. 83, 1–6 (2016). 93. Elleuche, S. Bringing functions together with fusion enzymes—from nature’s inventions to biotechnological applications. Applied Microbiology and Biotechnology 99, 1545–1556 (2015). 94. Patankar, S. C. & Yadav, G. D. Cascade Engineered Synthesis of γ-Valerolactone, 1,4-Pentanediol, and 2-Methyltetrahydrofuran from Levulinic Acid Using Pd-Cu/ZrO2Catalyst in Water as Solvent. ACS Sustain. Chem. Eng. 3, 2619–2630 (2015). 95. Ma, R., Yang, P., Ma, Y. & Bian, F. Facile Synthesis of Magnetic Hierarchical Core–Shell Structured Fe3O4@PDA-Pd@MOF Nanocomposites: Highly Integrated Multifunctional Catalysts. ChemCatChem 10, 1446–1454 (2018). 96. Yu, K., Liu, C., Kim, B. G. & Lee, D. Y. Synthetic fusion protein design and applications. Biotechnology Advances 33, 155–164 (2015). 97. Hailes, H. et al. Multi-step biocatalytic strategies for chiral amino alcohol synthesis. Enzyme Microb. Technol. 81, 23–30 (2015). 98. Both, P. et al. Whole-Cell Biocatalysts for Stereoselective C-H Amination Reactions. Angew. Chemie - Int. Ed. 55, 1511–1513 (2016). 99. Hartmann, M., Lee, S., Hallam, S. J. & Mohn, W. W. Bacterial, archaeal and eukaryal community structures throughout soil horizons of harvested and naturally disturbed forest stands. Environ. Microbiol. 11, 3045–3062 (2009). 100. Taupp, M., Lee, S., Hawley, A., Yang, J. & Hallam, S. J. Large Insert Environmental Genomic Library Production. J. Vis. Exp. (2009). doi:10.3791/1387 101. Wright, J. J., Lee, S., Zaikova, E., Walsh, D. a & Hallam, S. J. DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation. J. Vis. Exp. 3–6 (2009). doi:10.3791/1352 102. Lee, S. & Hallam, S. J. Extraction of High Molecular Weight Genomic DNA from Soils and Sediments. J. Vis. Exp. 2–5 (2009). doi:10.3791/1569 103. Finn, R. D., Clements, J. & Eddy, S. R. HMMER web server: Interactive sequence similarity searching. Nucleic Acids Res. 39, W29-37 (2011). 104. Alonso-Gutierrez, J. et al. Metabolic engineering of Escherichia coli for limonene and 174 perillyl alcohol production. Metab. Eng. 19, 33–41 (2013). 105. Cunningham, F. X., Sun, Z., Chamovitz, D., Hirschberg, J. & Gantt, E. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Plant Cell 6, 1107–1121 (1994). 106. Sasaki, K., Ohara, K. & Yazaki, K. Gene expression and characterization of isoprene synthase from Populus alba. FEBS Lett. 579, 2514–2518 (2005). 107. Kamiya, N., Kitayama, A., Ueda, H., Nagamune, T. & Arai, R. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng. Des. Sel. 14, 529–532 (2002). 108. Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews 65, 1357–1369 (2013). 109. Schwaneberg, U., Hamer, S. N., Cheng, F., Ruff, A. J. & Yang, J. Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli. Biotechnol. J. 11, 1639–1647 (2016). 110. Li, J., Li, Y., Cui, Z., Liang, Q. & Qi, Q. Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation. Appl. Microbiol. Biotechnol. 101, 3153–3161 (2017). 111. Rosano, G. L. & Ceccarelli, E. A. Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology 5, 172 (2014). 112. Albrecht, M., Misawa, N. & Sandmann, G. Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin. Biotechnol. Lett. 21, 791–795 (1999). 113. Lin, P. C. & Pakrasi, H. B. Engineering cyanobacteria for production of terpenoids. Planta 249, 145–154 (2018). 114. Xu, J. et al. Efficient production of lycopene by engineered E. coli strains harboring different types of plasmids. Bioprocess Biosyst. Eng. 41, 489–499 (2018). 115. Shen, H.-J., Hu, J.-J., Li, X.-R. & Liu, J.-Z. Engineering of Escherichia coli for Lycopene Production Through Promoter Engineering. Curr. Pharm. Biotechnol. 16, 1094–1103 (2015). 116. Quin, M. B., Wallin, K. K., Zhang, G. & Schmidt-Dannert, C. Spatial organization of multi-enzyme biocatalytic cascades. Org. Biomol. Chem. 15, 4260–4271 (2017). 117. Idan, O. & Hess, H. Origins of activity enhancement in enzyme cascades on scaffolds. ACS Nano 7, 8658–8665 (2013). 118. Microbiology, A. & Volke, D. Rational engineering of the methylerythritol 4-phosphate ( MEP ) pathway for terpenoid production through metabolic control analysis. 9, (2019). 119. Krasutsky, S. G. et al. Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens. J. Org. Chem. 79, 9170–9178 (2014). 120. ChemAxon - Software Solutions and Services for Chemistry & Biology. Available at: https://chemaxon.com/products/marvin. (Accessed: 4th March 2019) 121. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–61 (2010). 122. Homer, L. E. et al. Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae). Biochem. Syst. Ecol. 28, 367–382 (2000). 175 123. Rajeswara Rao, B. R., Kaul, P. N., Mallavarapu, G. R. & Ramesh, S. Effect of seasonal climatic changes on biomass yield and terpenoid composition of rose-scented geranium (Pelargonium species). Biochem. Syst. Ecol. 24, 627–635 (1996). 124. Waseem, R. & Low, K. H. Advanced analytical techniques for the extraction and characterization of plant-derived essential oils by gas chromatography with mass spectrometry. J. Sep. Sci. 38, 483–501 (2015). 125. Rajan, R., Chandran, K., Harper, S. L., Yun, S.-I. & Kalaichelvan, P. T. Plant extract synthesized silver nanoparticles: An ongoing source of novel biocompatible materials. Ind. Crops Prod. 70, 356–373 (2015). 126. Cavinder, C., Vogelsang, M., Gibbs, P., Forrest, D. & Scott, B. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Med. Aromat. Plants 04, 7 (2015). 127. Gallezot, P. Conversion of biomass to selected chemical products. Chem. Soc. Rev. 41, 1538–1558 (2012). 128. Liao, J. C., Mi, L., Pontrelli, S. & Luo, S. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 14, 288–304 (2016). 129. Sawisit, A., Jantama, S. S., Kanchanatawee, S. & Jantama, K. Efficient utilization of cassava pulp for succinate production by metabolically engineered Escherichia coli KJ122. Bioprocess Biosyst. Eng. 38, 175–187 (2015). 130. Edgar, S., Li, F.-S., Qiao, K., Weng, J.-K. & Stephanopoulos, G. Engineering of Taxadiene Synthase for Improved Selectivity and Yield of a Key Taxol Biosynthetic Intermediate. ACS Synth. Biol. acssynbio.6b00206 (2016). doi:10.1021/acssynbio.6b00206 131. Park, S. Y., Binkley, R. M., Kim, W. J., Lee, M. H. & Lee, S. Y. Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metab. Eng. 49, 105–115 (2018). 132. Diplom-Biochemiker Raimund Nagel, B., Gershenzon MPI für Chemische Ökologie, J., Hertweck Hans-Knöll-Institut, C. & Tissier, A. The regulatory function of isoprenyl diphosphate synthases in terpene biosynthesis in Halberstadt. (1983). 133. Renata, H., Wang, Z. J. & Arnold, F. H. Expanding the enzyme universe: Accessing non-natural reactions by mechanism-guided directed evolution. Angewandte Chemie - International Edition 54, 3351–3367 (2015). 134. Mafu, S., Sambandaswami, P., Ann, T. & Lee, B. Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase. 1–6 (2016). doi:10.1073/pnas.1612901114 135. Köksal, M., Jin, Y., Coates, R. M., Croteau, R. & Christianson, D. W. (Supp Info) Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis. Nature 469, 116–20 (2011). 136. Martin, D. M., Fäldt, J. & Bohlmann, J. Functional Characterization of Nine Norway SpruceTPS Genes and Evolution of GymnospermTerpene Synthases of the TPS-d Subfamily. Plant Physiol. 135, 1908–1927 (2004). 137. Köksal, M., Jin, Y., Coates, R. M., Croteau, R. & Christianson, D. W. Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis. Nature 469, 116–122 (2011). 138. Christianson, D. W. Unearthing the roots of the terpenome. Curr. Opin. Chem. Biol. 12, 141–150 (2008). 176 139. Li, Z. et al. The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene. Biochemistry 55, 6599–6604 (2016). 140. Srividya, N., Davis, E. M., Croteau, R. B. & Lange, B. M. Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase. Proc. Natl. Acad. Sci. U. S. A. 112, 3332–7 (2015). 141. Leferink, N. G. H. et al. Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome. ACS Catal. 8, 3780–3791 (2018). 142. Irmisch, S. et al. One amino acid makes the difference: the formation of ent-kaurene and 16α-hydroxy-ent-kaurane by diterpene synthases in poplar. BMC Plant Biol. 15, 262 (2015). 143. Wildung, M. R. & Croteau, R. A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. J. Biol. Chem. 271, 9201–9204 (1996). 144. Yadav, V. G. Unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase. J. Mol. Catal. B Enzym. 110, 154–164 (2014). 145. Bian, G. et al. Releasing the potential power of terpene synthases by a robust precursor supply platform. Metab. Eng. 42, 1–8 (2017). 146. Williams, D. C., McGarvey, D. J., Katahira, E. J. & Croteau, R. Truncation of limonene synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37, 12213–12220 (1998). 147. David C. Hyatt, Buhyun Youn, Yuxin Zhao, Bindu Santhamma, Robert M. Coates, Rodney B. Croteau, and C. K. Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc. Natl. Acad. Sci. 104, 5360–5365 (2006). 148. Whittington, D. A. et al. Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Proc. Natl. Acad. Sci. U. S. A. 99, 15375–80 (2002). 149. Bohlmann, J., Steele, C. L. & Croteau, R. Monoterpene synthases from grand fir (Abies grandis). cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase. J. Biol. Chem. 272, 21784–92 (1997). 150. Lodewyk, M. W., Gutta, P. & Tantillo, D. J. Computational studies on biosynthetic carbocation rearrangements leading to sativene, cyclosativene, α-ylangene, and β-ylangene. J. Org. Chem. 73, 6570–6579 (2008). 151. Fäldt, J., Martin, D., Miller, B., Rawat, S. & Bohlmann, J. Traumatic resin defense in Norway spruce (Picea abies): Methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase. Plant Mol. Biol. 51, 119–133 (2003). 152. Alonso-Gutierrez, J. et al. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metab. Eng. 19, 33–41 (2013). 153. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008). 154. Martin, D. M., Fäldt, J. & Bohlmann, J. Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol. 135, 1908–27 (2004). 177 155. Christianson, D. W. Structural and Chemical Biology of Terpenoid Cyclases. Chemical Reviews 117, 11570–11648 (2017). 156. Schrepfer, P. et al. Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases. Proc. Natl. Acad. Sci. 113, E958–E967 (2016). 157. Ferrara, M. A. et al. Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica. Brazilian J. Microbiol. 44, 1075–1080 (2013). 158. PubChem Search. Available at: https://pubchem.ncbi.nlm.nih.gov/search/. (Accessed: 14th February 2019) 159. Degenhardt, J., Köllner, T. G. & Gershenzon, J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70, 1621–1637 (2009). 160. Freeman, A., Woodley, J. M. & Lilly, M. D. In situ product removal as a tool for bioprocessing. Bio/Technology 11, 1007–1012 (1993). 161. Pollak, F. C. & Berger, R. G. Geosmin and Related Volatiles in Bioreactor-Cultured Streptomyces citreus CBS 109.60. Appl. Environ. Microbiol. 62, 1295–9 (1996). 162. Oda, S., Inada, Y., Kato, A., Matsudomi, N. & Ohta, H. Production of (S)-citronellic acid and (R)-citronellol with an interface bioreactor. J. Ferment. Bioeng. 80, 559–564 (1995). 163. Zhang, C. et al. Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae. Metab. Eng. 49, 28–35 (2018). 164. Covello, P. S. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528–532 (2013). 165. Pitera, D. J. et al. Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc. Natl. Acad. Sci. 109, E111–E118 (2012). 166. Srivastava, S. K. et al. A Biogenic Photovoltaic Material. Small 14, 1800729 (2018). 167. Grätzel, M. Dye-sensitized solar cells. Journal of Photochemistry and Photobiology C: Photochemistry Reviews 4, 145–153 (2003). 168. Ryan, M. Progress in ruthenium complexes for dye sensitised solar cells. Platin. Met. Rev. 53, 216–218 (2009). 169. Bella, F., Gerbaldi, C., Barolo, C. & Grätzel, M. Aqueous dye-sensitized solar cells. Chem. Soc. Rev. 44, 3431–3473 (2015). 170. Narayan, M. R. Review: Dye sensitized solar cells based on natural photosensitizers. Renewable and Sustainable Energy Reviews 16, 208–215 (2012). 171. Srivastava, S. K., Yamada, R., Ogino, C. & Kondo, A. Biogenic synthesis and characterization of gold nanoparticles by Escherichia coli K12 and its heterogeneous catalysis in degradation of 4-nitrophenol. Nanoscale Res. Lett. 8, 70 (2013). 172. Srivastava, S. K. & Constanti, M. Room temperature biogenic synthesis of multiple nanoparticles (Ag, Pd, Fe, Rh, Ni, Ru, Pt, Co, and Li) by Pseudomonas aeruginosa SM1. J. Nanoparticle Res. 14, 831–841 (2012). 173. Liu, L.-Y., Ji, L., Patankar, S., Yadav, V. & Ayakar, S. Isolation of phenolic monomers from kraft lignin using a magnetically recyclable TEMPO nanocatalyst. Green Chem. 21, 785–791 (2019). 174. Fargues, C., Mathias, Á. & Rodrigues, A. Kinetics of Vanillin Production from Kraft Lignin Oxidation. Ind. Eng. Chem. Res. 35, 28–36 (1996). 175. Bjørsvik, H. R. & Minisci, F. Fine chemicals from lignosulfonates. 1. Synthesis of vanillin by oxidation of lignosulfonates. Org. Process Res. Dev. 3, 330–340 (1999). 178 176. Rodrigues Pinto, P. C., Borges Da Silva, E. A. & Rodrigues, A. E. Insights into oxidative conversion of lignin to high-added-value phenolic aldehydes. in Industrial and Engineering Chemistry Research 50, 741–748 (2011). 177. Pacek, A. W., Ding, P., Garrett, M., Sheldrake, G. & Nienow, A. W. Catalytic conversion of sodium lignosulfonate to vanillin: Engineering aspects. part 1. effects of processing conditions on vanillin yield and selectivity. Ind. Eng. Chem. Res. 52, 8361–8372 (2013). 178. Schutyser, W. et al. Revisiting alkaline aerobic lignin oxidation. Green Chem. 20, 3828–3844 (2018). 179. Heaton, C. A. An Introduction to Industrial Chemistry. (Blackie Academic & Professional, 1991). 180. Valencia, R. The Future of the Chemical Industry by 2050. (Wiley-VCH Verlag GmbH & Co. KGaA., 2013). 181. Jones, M. E. Why big always wins: Examining economies of scale. Chemical Engineering (2013). 182. Patankar, S. C. & Renneckar, S. Greener synthesis of nanofibrillated cellulose using magnetically separable TEMPO nanocatalyst. Green Chem. 19, 4792–4797 (2017). 183. Klindworth, A. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, e1–e1 (2013). 184. White TJ, Bruns T, Lee S, T. J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. in PCR Protocols: A Guide to Methods and Applications (ed. Innis MA, Gelfand DH, Sninsky JJ, W. T.) 315–322 (New York: Academic Press Inc., 1990). 185. Coenye, T. et al. Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples. Int. J. Syst. Evol. Microbiol. 51, 1099–1107 (2001). 186. Stratilová, B. et al. Characterization of a long-chain α-galactosidase from Papiliotrema flavescens. World J. Microbiol. Biotechnol. 34, 19 (2018). 187. BacDive | The Bacterial Diversity Metadatabase. Available at: https://bacdive.dsmz.de/. (Accessed: 3rd March 2019) 188. Usov, I. et al. Understanding nanocellulose chirality and structure-properties relationship at the single fibril level. Nat. Commun. 6, 7564 (2015). 189. Peters, R. J., Croteau, R. B., Croteau, R. & Bohlmann, J. Abietadiene synthase catalysis: Mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement. Proc. Natl. Acad. Sci. 99, 580–584 (2002). 190. Vattekkatte, A. et al. Substrate geometry controls the cyclization cascade in multiproduct terpene synthases from Zea mays. Org. Biomol. Chem. 13, 6021–6030 (2015). 179 Appendices Appendix A Fusion protein sequences A.1 IspDF1 MIALQRSLSMHVTAIIAAAGEGRRLGAPLPKQLLDIGGRSILERSVMAFARHERIDDVIVVLPPALAAAPPDWIAASGRVPAVHVVSGGERRQDSVANAFDRVPAQSDVVLVHDAARPFVTAELISRAIDGAMQHGAAIVAVPVRDTVKRVDPDGEHPVITGTIPRDTIYLAQTPQAFRRDVLGAAVALGRSGVSATDEAMLAEQAGHRVHVVEGDPANVKITTSADLDQARQRLRSAVAARIGTGYDLHRLIEGRPLIIGGVAVPCDKGALGHSDADVACHAVIDALLGAAGAGNVGQHYPDTDPRWKGASSIGLLRDALRLVQERGFTVENVDVCVVLERPKIAPFIPEIRARIAGALGIDPERVSVKGKTNEGVDAVGRGEAIAAHAVALLSES A.2 IspDF2 MRCEIPHRCVRRKYRIRRHSPFRLRNCWEGRSVRASSLLRQLAKGGCAARRLSWVTPGFSQSRRCKTTASELTRIGIGIRIQGDIMQVTAIIAAGGRGRRFGGGVPKQLVGVGGRPILERTVAAFLGHPAIHEVVVALPAELMADPPAYLRAAPKPIRLVAGGVQRQDSVRQAFQAANEQSDVIVIHDAARPFASADLISRTIAAAAEGGAALAAVPARDTVKRGAFAAGRTGPAGRQAVEGAPLLVVAETLPRDSIYLAQTPQAFRRDVLRDALALGEAGSEATDEATLAERAGHIVRLVEGEPANIKITTPDDLLVAEAIARGTGERAVGERAAFRIGAGYDLHRLVEGRPLVLGGVTIPFERGLLGHSDADAICHAVTDAVLGAAAAGDIGRHFPDSDPKWRDWSSIDLLRRASAIVKGRGYAIANVDAVVIAERPKLAPFLDEMRANVAGAIGIAVDAVGIKGKTNEGLGELGRGEAIAVHAVALLHL 180 A.3 IspDF3 MRCEIPHRCVRRKYRIRRHSPFRLRNCWEGRSVRASSLLRQLAKGGCAARRLSWVTPGFSQSRRCKTTASELTRIGIGIRIQGDIMVHVSAIIAAGGRGERFGGPQPKQLLLLGGVPILKRTVDAFLRGYPFIEVIVALPAEFVANPPDYLDDVIVVEGGARRQDSVANAFRAVAPSAQVVVIHDAARPLVTPSLIERTVDAAVKHGAAIAALRATDTVKRGDASRVIRGTLPRDEIFLAQTPQAFRAGVLRDALALAASAADATDEAMLAEQAGHHVRLVDGDPRNLKITTPEDLEMAERLIGARNTAGAMRIGNGYDLHRLVTGRPLVLGGVTIPFEKGLQGHSDADAVCHAITDAILGAASAGDIGRHFPDTDPAWKDAKSIVLLQQAAQIVSRAGYAIANLDVVVIAQQPKLVPHIDAIRHSVAHALGIDVQQVSVKGKTNEGVDSMGAGESIAVHAVALLQHS 181 Appendix B HPLC chromatograms for ATP, ADP and AMP analysis Figure 0-1 HPLC chromatogram for set 2 IspE in vitro reactions. The peaks for ATP (2.49 min), ADP (2.60 min) and AMP (2.72 min) are labelled. 182 Appendix C GC-MS analysis for terpenes Figure 0-2 GC-MS Chromatograms of aerobic shake flask fermentation analysis for SACar, SAMyr, SALin and SALim+. The monoterpene retention times are: pinene 6.48 min, sabinene 7.42 min, myrcene 7.86 min, carene 8.42 min, limonene 8.88 min, ocimene 9.36 min, terpinene 9.61 min, terpinolene 10.20 min, linalool 10.35 min.
Thesis/Dissertation
eng
Chemical and Biological Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Biocatalysis and bioprocess engineering for terpenoid production
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