Science, Faculty of
DSpace
UBCV
Sunstrum, Frederick Guy
Master of Science - MSc
University of British Columbia
The plant kingdom has always been a useful source of therapeutic molecules for humans. Montbretin A (MbA) is a complex acylated flavonol glycoside found in the underground storage organs, called corms, of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA was discovered as a highly efficient and specific inhibitor of the human pancreatic α-amylase (HPA), making it an attractive candidate for the treatment of type 2 diabetes, a worldwide epidemic. The original plant source montbretia is not adapted for large scale cultivation, and MbAâs complex structure renders chemical synthesis infeasible for scalable production. Therefore, we are aiming to engineer a heterologous system for large-scale sustainable MbA production for treatment of type 2 diabetes. To this end we are using synthetic biology to develop an MbA producing Nicotiana benthamiana (tobacco) strain. This is possible due to the recent complete elucidation of the MbA biosynthetic pathway, including all essential genes and enzymes from montbretia. Small amounts of MbA can be produced in N. benthamiana through transient expression of the MbA biosynthetic enzymes. However, much higher quantities of montbretin B (MbB) and montbretin C (MbC), which differ from MbA in the identity of their acyl groups, were produced. MbB and MbC possess a coumaroyl and a feruloyl group, respectively, and are not effective HPA inhibitors. To influence acyl group availability, specifically increase the amount of caffeoyl-CoA for MbA biosynthesis, we investigated a set of enzymes involved in caffeoyl-CoA biosynthesis in montbretia, specifically 4-coumaroyl CoA ligases (4CL), hydroxycinnamoyl- CoA shikimate/quinate hydroxycinnamoyltransferases (HCT), 4-coumarate 3-hydroxylases (C3Hs), and caffeoyl-shikimate esterases (CSE). Co-expression of those caffeoyl-CoA biosynthetic genes together with MbA biosynthetic genes resulted in up to 10-fold higher MbA yields.
ENGINEERING MONTBRETIN A BIOSYNTHESIS IN NICOTIANA BENTHAMIANA by Frederick Guy Sunstrum B.Sc., The University of Ottawa, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Genome Science and Technology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2021 © Frederick Guy Sunstrum, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Engineering Montbretin A Biosynthesis in Nicotiana benthamiana submitted by Frederick Guy Sunstrum in partial fulfillment of the requirements for the degree of Master of Science in Genome Science and Technology Examining Committee: Dr. Jörg Bohlmann, Professor, Michael Smith Laboratories, UBC Supervisor Dr. Stephen Withers, Professor, Michael Smith Laboratories, UBC Supervisory Committee Member Dr. Vikramaditya Yadav, Professor, Chemical and Biological Engineering, UBC Supervisory Committee Member Dr. Robert Guy, Professor, Forestry, UBC Additional Examiner iii Abstract The plant kingdom has always been a useful source of therapeutic molecules for humans. Montbretin A (MbA) is a complex acylated flavonol glycoside found in the underground storage organs, called corms, of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA was discovered as a highly efficient and specific inhibitor of the human pancreatic α-amylase (HPA), making it an attractive candidate for the treatment of type 2 diabetes, a worldwide epidemic. The original plant source montbretia is not adapted for large scale cultivation, and MbAâs complex structure renders chemical synthesis infeasible for scalable production. Therefore, we are aiming to engineer a heterologous system for large-scale sustainable MbA production for treatment of type 2 diabetes. To this end we are using synthetic biology to develop an MbA producing Nicotiana benthamiana (tobacco) strain. This is possible due to the recent complete elucidation of the MbA biosynthetic pathway, including all essential genes and enzymes from montbretia. Small amounts of MbA can be produced in N. benthamiana through transient expression of the MbA biosynthetic enzymes. However, much higher quantities of montbretin B (MbB) and montbretin C (MbC), which differ from MbA in the identity of their acyl groups, were produced. MbB and MbC possess a coumaroyl and a feruloyl group, respectively, and are not effective HPA inhibitors. To influence acyl group availability, specifically increase the amount of caffeoyl-CoA for MbA biosynthesis, we investigated a set of enzymes involved in caffeoyl-CoA biosynthesis in montbretia, specifically 4-coumaroyl CoA ligases (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases (HCT), 4-coumarate 3-hydroxylases (C3Hs), and caffeoyl-shikimate esterases (CSE). Co-expression of those caffeoyl-CoA iv biosynthetic genes together with MbA biosynthetic genes resulted in up to 10-fold higher MbA yields. v Lay Summary Montbretin A (MbA) is promising as a potential new treatment for type 2 diabetes. The molecule MbA is produced by the ornamental plant montbretia in below ground storage organs. However, if MbA is approved as a type 2 diabetes treatment, agricultural production of montbretia or chemical synthesis are not expected to satisfy expected demand for the compound. An alternative production method is by engineering another organism such as tobacco to produce MbA. With this goal in mind, the main genes and enzymes involved in the production of MbA have been identified, which has enabled the production of small amounts of the compound in engineered tobacco. This thesis aims to improve MbA yields in this system, specifically by improving supply of the key precursor caffeoyl-CoA. By co-expressing genes involved in caffeoyl-CoA production with those required for MbA production in tobacco, MbA yields were improved up to 10-fold. vi Preface Chapter 2 Chapter 2 has been accepted for publication. I conducted cloning, protein expression, enzyme assays, and transient expression in N. benthamiana with assistance from Sandra Irmisch and Sharon Jancsik. Lina Madilao assisted with liquid chromatography and mass spectrometry analyses. Hannah Liu, Sandra Irmisch, and Sharon Jancsik reconstructed flavanone biosynthesis in Saccharomyces cerevisiae. Mack Yuen provided advice with differential expression analysis. The manuscript was written by me alongside Sandra Irmisch and Jörg Bohlmann. Sunstrum, Frederick G., Liu, Hannah L., Jancsik, Sharon, Madilao, Lufiani L. 2021. â4-coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin Aâ PLOS ONE. 16: 1-18. doi:10.1371/journal.pone.0257478 Chapter 3 I conducted cloning, protein expression, enzyme assays, and transient expression in N. benthamiana with assistance from Sandra Irmisch and Sharon Jancsik. Lina Madilao assisted with liquid chromatography and mass spectrometry analyses. Sandra Irmisch assisted with gene selection and primer design. Mack Yuen provided support with the metabolomic data analysis. Lars Kruse provided enzymatic products for use as analytical standards for caffeoyl-shikimate, coumaroyl-shikimate, and coumaroyl-quinate. Chapter 3 was written by me and reviewed by Jörg Bohlmann. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Figures ............................................................................................................................... xi Acknowledgements .................................................................................................................... xiii Chapter 1: Introduction ................................................................................................................1 1.1 Plant specialized metabolism .......................................................................................... 1 1.2 Diabetes and Montbretin A ............................................................................................. 3 1.3 Objectives ....................................................................................................................... 6 Chapter 2: 4-coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A ..................................................................................................................................7 2.1 Summary ......................................................................................................................... 7 2.2 Introduction ..................................................................................................................... 8 2.3 Materials and Methods .................................................................................................. 12 2.3.1 Identification of target CcAAE .............................................................................. 12 2.3.2 Cloning and heterologous expression in E. coli .................................................... 13 2.3.3 Enzyme assays with recombinant CcAAEs .......................................................... 14 2.3.4 Transient expression of CcAAEs in Nicotiana benthamiana ............................... 15 2.3.5 Reconstruction of flavanone biosynthesis in yeast ............................................... 16 2.3.6 LC-MS analyses .................................................................................................... 17 viii 2.3.7 Statistical analysis ................................................................................................. 18 2.3.8 Accession Numbers .............................................................................................. 18 2.4 Results ........................................................................................................................... 18 2.4.1 Identification of AAEs expressed in montbretia corms ........................................ 18 2.4.2 Cc4CL1 and Cc4CL2 activate hydroxycinnamic acids ........................................ 21 2.4.3 CcAAEs did not affect MbA levels when transiently expressed in N. benthamiana â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦24 2.4.4 Utilizing Cc4CLs to reconstruct flavanone biosynthesis in Saccharomyces cerevisiae .............................................................................................................. 25 2.5 Discussion ..................................................................................................................... 27 Chapter 3: Improving montbretin A yields in engineered Nicotiana benthamiana ...............32 3.1 Summary ....................................................................................................................... 32 3.2 Introduction ................................................................................................................... 33 3.3 Materials and Methods .................................................................................................. 37 3.3.1 Cloning .................................................................................................................. 37 3.3.2 Transient expression in N. benthamiana ............................................................... 38 3.3.3 LC-MS analyses .................................................................................................... 40 3.3.4 Untargeted metabolomics analyses ....................................................................... 40 3.4 Results ........................................................................................................................... 41 3.4.1 Effects of phenylpropanoid biosynthetic genes on montbretin levels in engineered N. benthamiana ..................................................................................................................... 41 3.4.2 Confirmation of HCT3 and CSE2 activities with the complete MbA pathway ... 43 3.4.3 Ustilago maydis chlorogenic acid esterase ........................................................... 44 ix 3.4.4 Substrate feeding ................................................................................................... 45 3.4.5 Metabolomics ........................................................................................................ 46 3.5 Discussion ..................................................................................................................... 50 Chapter 4: Conclusion .................................................................................................................54 Bibliography .................................................................................................................................58 Appendices ....................................................................................................................................73 Appendix A ............................................................................................................................... 73 A.1 Protein accession numbers. ....................................................................................... 73 A.2 Contig names and PTS1 tripeptide if present. .......................................................... 73 A.3 Correlation of CcAAEs expression with previously identified MbA pathway genes during young corm development. ......................................................................................... 75 A.4 Expression data of 16 candidate AAEs. .................................................................... 76 A.5 Activity and expression of AAEs. ............................................................................ 77 A.6 Combinations of genes used for transient expression in N. benthamiana ................ 78 A.7 Montbretin A, B and C levels in N. benthamiana plants transiently expressing MBGs and AAE. ................................................................................................................... 79 A.8 Identification of montbretins produced by N. benthamiana transiently expressing MBGs.. .................................................................................................................................. 80 A.9 Parts of the MoClo system (Addgene, MA, USA) used for the construction of S. cerevisiae expression modules. ............................................................................................. 81 A.10 Identification of products formed by S. cerevisiae expressing a flavanone biosynthetic module.. ............................................................................................................ 81 A.11 Oligonucleotides used in this study. ......................................................................... 82 x Appendix B ............................................................................................................................... 83 B.1 Montbretia phenylpropanoid biosynthesis genes ...................................................... 83 B.2 Optimal parameters for xcms as determined by the âIPOâ package. ....................... 84 B.3 Additional combinations of genes used for transient expression in N. benthamiana. â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦85 xi List of Figures Figure 1.1 Structures and inhibitor activity of montbretin A, B and C found in montbretia corms.......................................................................................................................................................... 4 Figure 2.1 Phylogeny of montbretia acyl activating enzymes (AAEs). ....................................... 19 Figure 2.2 Temporal expression profile of montbretia AAEs and previously characterized MbA biosynthetic genes. ........................................................................................................................ 21 Figure 2.3 Enzyme activities of Cc4CL1 and Cc4CL2. ............................................................... 23 Figure 2.4 Montbretin accumulation in N. benthamiana co-expressing MbA biosynthetic genes and Cc4CL1 or Cc4CL2. .............................................................................................................. 25 Figure 2.5 Reconstruction of flavanone biosynthesis in Saccharomyces cerevisiae using montbretia 4CLs. .......................................................................................................................... 26 Figure 3.1 General phenylpropanoid pathway. Adapted from Vanholme et al., 2013. ................ 36 Figure 3.2 Intended reaction for UmChlE expression in N. benthamiana. ................................... 37 Figure 3.3 Screening phenylpropanoid biosynthetic genes for improvements in mini-MbA yields in Nicotiana benthamiana. ............................................................................................................ 42 Figure 3.4 Montbretin accumulation in N. benthamiana co-expressing mini-MbA biosynthetic genes and various phenylpropanoid biosynthetic genes. .............................................................. 43 Figure 3.5 Montbretin accumulation in N. benthamiana co-expressing MbA biosynthetic genes and a chlorogenic acid esterase from Ustilago maydis (UmChlE). .............................................. 45 Figure 3.6 Substrate feeding experiment in N. benthamiana co-expressing mini-MbA biosynthetic genes (MBGs) and both montbretia 4CLs. ............................................................... 46 Figure 3.7 Metabolic changes induced by expression of CcCSE2, CcHCT3, and UmChlE. ....... 47 xii Figure 3.8 Abundance of intermediates in caffeoyl-CoA biosynthesis in N. benthamiana plants expressing MBGs and genes improving MbA yields. .................................................................. 49 xiii Acknowledgements Special thanks to Dr. Sandra Irmisch, without whom this project would not have been possible. Many thanks to Dr. Jörg Bohlmann for his support throughout my studies. I also thank all the friends, colleagues, and family who kept me going over the years. 1 Chapter 1: Introduction 1.1 Plant specialized metabolism The plant kingdom is a source of diverse compounds used in human diet and medicine. The sessile nature of plants requires them to depend on a variety of bioactive molecules to survive and interact with their environment. These specialized metabolites generally have limited taxonomic distributions, are not commonly involved in growth or energy metabolism, and generally have functions adapted to the specialized niches of their respective species. Plants are estimated to produce over 200,000 unique chemical structures (Tohge, Scossa, and Fernie 2015; Dixon and Strack 2003; Yonekura-Sakakibara and Saito 2009). This chemical diversity is the source of a high proportion of approved pharmaceutical drugs (Newman and Cragg 2020). With the advent of modern high-throughput screening techniques there is a renewed interest in exploiting chemical diversity in plants for the discovery and development of therapeutic compounds. Plant specialized metabolites are separated into three large categories based on their biosynthetic origins, plus several other groups of metabolites. Phenolics are produced in the phenylpropanoid pathway and originate from aromatic amino acids. Many phenolic compounds are widespread throughout the plant kingdom and have important functions such as UV protection, antioxidants, pest resistance, and structural integrity in the case of lignin (Vogt 2010). Terpenoids are a diverse group of metabolites derived from 5-carbon monomers (Pichersky and Raguso 2018; F. Chen et al. 2011). Finally, alkaloids are nitrogen containing metabolites often derived from amino acids. The diversity, complexity, and scarcity of metabolites produced by plants can make large scale production difficult. When chemical synthesis is not a feasible source, metabolic 2 engineering has emerged as an alternative production method. This approach involves introducing genes involved in the biosynthesis of a compound of interest into a heterologous host. For example, Taxol is a leading cancer drug discovered in the bark of Pacific yew (Taxus brevifolia), whoâs increased demand led to risk of widespread deforestation of yew (Cragg et al. 1993). Today, Taxol or its precursors can be produced using Taxus cell cultures, engineered yeast or tobacco (Engels, Dahm, and Jennewein 2008; Fett-neto et al. 1992; J. Li et al. 2019). The difficulty in securing a reliable source of Taxol underlined the importance of securing sustainable sources for plant-sourced medicines. Even in cases where agricultural production exists, establishing a complementary source of a drug can be beneficial to secure and stabilize supply chains. Production of artemisinin, a component in a leading malaria drug produced by Artemisia annua, struggled with inconsistent harvests combined with increased demand (Ikram and Simonsen 2017). Geopolitical concerns regarding opium poppy cultivation motivated the development of engineered yeast for opioid production (Galanie et al. 2015). A synthetic biology approach to produce complex plant metabolites has several advantages relative to chemical synthesis or agricultural production. Inputs can be as simple as glucose for microbes, or CO2 and water for photosynthetic organisms. Common hosts for metabolic engineering include E. coli, yeast (Saccharomyces cerevisiae), or wild tobacco (Nicotiana benthamiana). N. benthamiana holds some advantages over microbial species for the production of complex plant specialized metabolites: it possesses the necessary cellular machinery for the expression of plant enzymes, produces many precursors absent in microbes thus requiring fewer engineered biosynthetic steps, and is easily transformed using Agrobacterium tumefaciens. 3 1.2 Diabetes and Montbretin A Type II diabetes is a chronic disease characterized by high blood glucose levels, and is a major cause of blindness, kidney failure, and heart disease. The disease was estimated to affect over 450 million people worldwide in 2017 and projected to increase to 693 million by 2045 (Cho et al. 2018). Diabetes treatment typically involves a combination of lifestyle changes, and insulin or non-insulin drugs. Three currently employed drugs, acarbose, miglitol, and voglibose, target enzymes involved in starch digestion resulting in lower blood sugar levels following a meal. Starch digestion is primarily initiated by human pancreatic α-amylase (HPA) yielding oligosaccharides, which are then digested into glucose by various α-glucosidases. Acarbose, miglitol, and voglibose all act as α-glucosidase inhibitors, and acarbose also targets HPA. These drugs also have side-effects such as abdominal pain, flatulence, and diarrhea, which can result in poor patient compliance (Chiasson et al. 1994; García-Pérez et al. 2013). These side effects are caused by the undigested oligosaccharides undergoing anaerobic fermentation in the lower gut. A specific HPA inhibitor that doesnât inhibit α-glucosidases should inhibit starch digestion and lower blood sugar without these negative side-effects. In 2008, montbretin A (MbA) was identified as a potent and specific (Ki = 8.1 nM) HPA inhibitor in a large-scale screen of 30,000 natural product extracts of plant and marine origin (Tarling et al. 2008). MbA was purified from the belowground storage organs of Crocosmia x crocosmiiflora (Asada, Hirayama, and Furuya 1988). This ornamental plant from the Iridaceae family is commonly referred to as montbretia. MbAâs effectiveness in reducing post-prandial blood glucose levels has been demonstrated in a diabetic rat model (Yuen et al. 2016). MbA is a complex acylated flavonol glycoside, defined as myricetin 3-O-(glucosyl-6â-O-caffeoyl)-1,2-Î²-D-glucosyl 1,2-α-L-rhamnoside 4â-O-Î±-L-rhamnosyl 1,4-β-D-xyloside. Two related compounds, 4 called MbB and MbC, which respectively possess a coumaroyl and feruloyl in the place of MbAâs caffeoyl, also occur in montbretia corms (Asada, Hirayama, and Furuya 1988; Figure 1.1). However, they are not efficient HPA inhibitors (Williams et al. 2015). The high molecular weight and polarity of MbA should minimize absorption into the blood stream, which is advantageous for a drug with its site of action in the gut. Figure 1.1 Structures and inhibitor activity of montbretin A, B and C found in montbretia corms (A) Montbretin A (MbA) contains a caffeoyl moiety which contributes to the highly efficient inhibition of human pancreatic α-amylase (HPA). MbB and MbC have a coumaroyl- and feruloyl moiety, respectively, and are not effective HPA inhibitors. (B) Ki values for HPA inhibition were reported previously (Williams et al., 2015). MbAâs highly complex structure eliminates chemical synthesis as a viable source of the compound. In addition, montbretia is the only known source of MbA. Montbretia is not adapted for large-scale cultivation, and MbA occurs in montbretia corms which are involved in R Ki (nM)Montbretin A OH 8.1 ± 0.8Montbretin B H 3600 ± 100Montbretin C OCH3 6100 ± 100ABR5 vegetative reproduction. Thus, montbretia plants are not considered to be sufficient to produce industrial amounts of MbA. In the event of MbA being approved for clinical use, an alternative source of MbA will be required to solve the supply problem. The difficulties of a reliable and scalable source of MbA make it an interesting candidate for metabolic engineering in N. benthamiana. For this approach to be successful, in-depth knowledge of MbA biosynthesis is required. MbA biosynthesis was determined to primarily occur in newly developing corms, early in the growing season (Irmisch et al. 2018; 2020). By comparing the temporal expression pattern of candidate montbretin biosynthetic genes (MBGs) to MbA accumulation in corms, five different UDP-dependent glycosyltransferases and one BAHD acyltransferase were identified and found to catalyze the sequential assembly of MbA from myricetin, two units of UDP-rhamnose, two units of UDP glucose, one unit of UDP-rhamnose, and one unit of caffeoyl-CoA (Irmisch et al. 2020; 2018; Irmisch, Jancsik, et al. 2019). The third step of MbA biosynthesis is the acylation of myricetin 3-O-glucosyl rhamnoside (MRG) with caffeoyl-CoA catalyzed by CcAT to generate myricetin 3-O-(6â-O-caffeoyl)-glucosyl rhamnoside (mini-MbA; Irmisch et al. 2018). CcAT also has the ability to accept coumaroyl-CoA and feruloyl-CoA to generate mini-MbB and mini-MbC, respectively. A set of genes involved in myricetin biosynthesis have also been identified (Irmisch, Ruebsam, et al. 2019). The discovery of these montbretin biosynthetic genes has enabled production of MbA in N. benthamiana by Agrobacterium-mediated transient expression (Irmisch et al. 2020). However, less than 10 µg/g of MbA is produced in transformed leaves, which is surpassed by much higher levels of MbB and MbC. The fact that CcAT can accept multiple substrates suggests that product identity is determined by substrate availability. Thus, MbA yields could be improved by 6 increasing the availability of caffeoyl-CoA. The fact that MbB is produced in large amounts indicates that p-coumaroyl-CoA is available, and that this pool of p-coumaroyl-CoA could be converted to caffeoyl-CoA. 1.3 Objectives The overall goal of this thesis is to improve the metabolic engineering of MbA in N. benthamiana. The specific objective of my thesis is to identify genes of MbA biosynthesis that direct the formation of predominantly MbA over the weak HPA inhibitors MbB and MbC. My underlying hypothesis is that substrate availability of caffeoyl-CoA over coumaroyl-CoA is a key factor in selecting MbA production over that of MbB. Co-expressing genes involved in the conversion of coumaroyl-CoA to caffeoyl-CoA with montbretin biosynthetic genes in N. benthamiana should result in increased MbA production. 7 Chapter 2: 4-coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin A 2.1 Summary Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis. Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that 8 both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae). Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast. 2.2 Introduction The large chemical space of specialized (i.e., secondary) metabolites produced by plants contains some of the most powerful medicinally active compounds. Montbretia (Crocosmia x crocosmiiflora), a member of the Iris family (Iridaceae), produces the flavonoid molecule montbretin A (MbA) and a series of related molecules MbB and MbC (Asada, Hirayama, and Furuya 1988; Tarling et al. 2008) (Figure 2.1). MbA is a complex acylated flavonol glycoside, defined as myricetin 3-O-(glucosyl-6â-O-caffeoyl)-1,2-Î²-D-glucosyl 1,2-α-L-rhamnoside 4â-O-Î±-L-rhamnosyl 1,4-β-D-xyloside. MbA inhibits human pancreatic α-amylase (HPA) in a highly efficient and selective manner, thereby slowing starch digestion in the intestine and reducing post-prandial blood glucose levels (Yuen et al. 2016). This unique activity and specificity make MbA a candidate for its development into a novel pharmaceutical or nutraceutical to treat or prevent type-2 diabetes and obesity (Tarling et al. 2008; Williams et al. 2015). MbA has successfully passed animal trials with diabetic rats and is now entering human clinical trials (Yuen et al. 2016). A sustainable and scalable production system is needed to develop MbA as a 9 widely available pharmaceutical or nutraceutical. Currently, the only known source for MbA are the underground storage and reproductive organs, called corms, of montbretia (Irmisch et al. 2018). The rare taxonomic occurrence and low amounts of MbA in montbretia corms (Tarling et al. 2008; Irmisch et al. 2018) together with the lack of large-scale cultivation, currently render montbretia inadequate for farm-based MbA production. Large-scale chemical synthesis of MbA is also not feasible due to its complex chemical structure. MbA production by means of synthetic biology or metabolic engineering of a recombinant plant or microbial system appears to be a viable approach (Irmisch et al. 2018; Irmisch, Ruebsam, et al. 2019; Irmisch, Jancsik, et al. 2019; Irmisch et al. 2020). To enable metabolic engineering of MbA production, we are investigating the genes and enzymes of MbA biosynthesis in montbretia. We recently elucidated the assembly of the complete MbA molecule from its different building blocks, specifically myricetin, UDP-glucose, UDP-rhamnose, UDP-xylose, and caffeoyl-CoA. Five UDP-dependent glycosyltransferases (UGTs) and a BAHD-acyltransferase (AT) have been identified to catalyze MbA assembly in a linear six-step biosynthetic pathway (Irmisch et al. 2018; 2020; Irmisch, Jancsik, et al. 2019). The third step in MbA biosynthesis is the acylation of myricetin glycosyl rhamnoside (MRG) into mini-MbA catalyzed by CcAT. This enzyme is not specific for its acyl donor and accepts different hydroxycinnamoyl-CoA thioesters as substrates. The incorporation of caffeoyl-CoA, coumaroyl-CoA, or feruloyl-CoA leads to the formation of MbA, montbretin B (MbB), or montbretin C (MbC), respectively (Figure 2.1). The meta-hydroxy group of the caffeoyl moiety in MbA is essential for the biological activity of HPA inhibition; MbB and MbC are not efficient inhibitors of HPA due to the absence and methylation, respectively, of this meta-hydroxy group (Tarling et al. 2008; Williams et al. 2015). 10 We are exploring both yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana as hosts for metabolic engineering of MbA production. Successful reconstruction of the MbA biosynthetic pathway was achieved in N. benthamiana through transient expression of MbA assembly genes together with montbretia flavonol biosynthesis genes (Irmisch, Ruebsam, et al. 2019; Irmisch et al. 2020). While MbA is the predominant montbretin in montbretia corms, transiently transformed N. benthamiana predominantly produced MbB likely due to better availability of coumaroyl-CoA relative to caffeoyl-CoA. The hydroxycinnamoyl-CoA thioesters caffeoyl-CoA, coumaroyl-CoA and feruloyl-CoA are produced from the respective hydroxycinnamic acids by enzymes generally referred to as 4-coumaroyl-CoA ligases (4CLs) (Cukovic et al. 2001; Y. Li et al. 2015). 4CLs belong to the larger family of acyl-activating enzymes (AAEs), which mainly catalyze the conjugation of an organic acid to CoA using an ATP-dependent two-step mechanism (Shockey and Browse 2011). The AAE family can be divided into seven clades with 4CLs clustering in clade IV. However, enzymes possessing 4CL activity have also been identified in clades V, VI, and VII (Gaid et al. 2012; Klempien et al. 2012; S. Lee et al. 2012; Martin et al. 2016; Schnabel et al. 2019; Srividya et al. 2020). As enzymes in the central phenylpropanoid pathway, 4CLs are ubiquitous across the plant kingdom (Gao et al. 2012; Lavhale, Kalunke, and Giri 2018). They are best known for the production of coumaroyl-CoA which serves as a precursor for a multitude of metabolites, including lignin and flavonoids. AAE and specifically 4CLs have not been explored in montbretia, where they are likely involved in two different biosynthetic reactions essential for MbA biosynthesis, the production of coumaroyl-CoA for the biosynthesis of the core flavonol myricetin and the production of caffeoyl-CoA as the acyl donor for CcAT in MbA assembly. In this work we explore the 11 molecular basis for the formation of hydroxycinnamoyl-CoA precursors for myricetin and MbA production in montbretia and the utility of Cc4CLs for metabolic engineering. We describe the discovery and characterization of two montbretia 4CLs and their heterologous expression in N. benthamiana and S. cerevisiae in the context of MbA production. 12 2.3 Materials and Methods 2.3.1 Identification of target CcAAE RNA extraction from montbretia corms, cDNA synthesis, and corm time-course transcriptome sequencing, assembly, and differential expression analysis was described previously (Irmisch et al. 2018; 2020). To identify putative AAEs involved in MbA biosynthesis, a BLASTP search of the translated montbretia transcriptome was conducted using a set of characterized AAE (Appendix A.1). Results were filtered using a reciprocal BLASTP search against the non-redundant protein database of NCBI. Resulting montbretia AAEs were used in a second reciprocal BLASTP analysis. Only sequences clustering into clade IV, V, VI and VII of the AAE family were considered as candidates. This process resulted in 38 putative AAEs. Sixteen of those were longer than 400 amino acids and retained for further analysis. Transcript expression data in counts per million (cpm), from six different developmental time-points of montbretia young corms, were generated as previously described (Irmisch et al. 2020). We used this time-course expression data to compute Pearson correlation coefficients (Appendix A.3). Genes which clustered into the 4CL clade and/or showed a correlation coefficient > 0.9 were selected yielding eight final candidate genes. Expression data for 16 AAE can be found in Appendix A.4. A heatmap visualizing expression patterns of putative CcAAEs alongside characterized montbretia myricetin and MbA biosynthetic genes was generated in R 4.0.2 (R Core Team, 2020) using the âpheatmapâ package (Kolde 2015). Genes possessing similar transcript expression patterns were determined by hierarchical clustering using Pearson correlation distance and complete-linkage clustering. Each data point represents normalized mean counts per million from two biological replicates. 13 The predicted amino acid sequences of putative montbretia AAEs and AAE from other plants were aligned using the ClustalW algorithm and the GONNET protein weight matrix implemented in MEGA7 (Kumar, Stecher, and Tamura 2016) (Appendix A1). This alignment was used to generate a phylogenetic tree with MEGA7 using the neighbour-joining algorithm and the Poisson model. The tree topology was evaluated by performing a bootstrap resampling analysis with 1000 replicates. 2.3.2 Cloning and heterologous expression in E. coli The eight selected CcAAEs were amplified from young corm cDNA (harvested June 10th, 2016) and cloned into the pJet1.2/blunt vector for sequencing (Appendix A.11) (Irmisch et al. 2018). Complete open reading frames of the target CcAAEs were cloned as BsaI or BbsI fragments into the pASK-IBA37+ expression vector (IBA), for N-terminal His6-tagging. The E. coli TOP10 strain (Invitrogen) was used for heterologous CcAAE expression. Cultures were grown at 21°C, 220 rpm and induced at OD600 = 0.6 with 200 μg/L anhydrotetracycline (Sigma-Aldrich) and further cultured at 18°C for 20 h. Cultures were collected by centrifugation (4300 x g) and disrupted by five freeze and thaw cycles in chilled extraction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 10% [v/v] glycerol, 1x PierceTM protease inhibitor [EDTA-free; Thermo Fisher Scientific], 25 U Benzonase Nuclease [Merck, Germany], and 0.2 mg/mL lysozyme). Cell fragments were removed by centrifugation (14 000 x g), and the supernatant was desalted into assay buffer (10 mM Tris-HCl, pH 7.5, 1 mM DTT, and 10% [v/v] glycerol) using Econopac 10DG columns (Bio-Rad, USA). For protein purification of 4CL1 and 4CL2 using Ni-NTA, a modified extraction buffer was used (50mM Tris-HCl, pH 7.5; 10mM MgCl2;5mM DTT; 2% [v/v] glycerol; 150 mM NaCl2; 20 mM imidazole; 1x Pierce protease inhibitor [EDTA-14 free, ThermoFisher Scientific], 25 U Benzonase Nuclease [Merck, Germany], and 0.2 mg/mL lysozyme) and the lysate was directly loaded onto a Ni-NTA agarose column (Qiagen). Protein was eluted with elution buffer (10 mM Tris-HCl, pH 7.5; 400 mM imidazole; 1mM DTT; 10% [v/v] glycerol) and desalted into assay buffer using Illustra NAP-5 Columns (GE Healthcare). Enzyme assays were set up the same day as storage at 4°C or shock-freezing of the purified protein led to loss of activity. The Monoclonal AntipolyHistidine-Alkaline Phosphatase antibody (Sigma-Aldrich) and the 1-Step NBT/BCIP Substrate Solution (Thermo Fisher Scientific) were used to ensure successful heterologous enzyme expression (Appendix A.5B). 2.3.3 Enzyme assays with recombinant CcAAEs To test for 4CL activity, initial enzyme assays were performed with 100 μL bacterial extract, 5 mM ATP, 5 mM MgCl2, 200 μM CoA (Sigma-Aldrich, Germany), and 100 μM of substrate in a Teflon-sealed, screw-capped 1 mL glass vial for 2 h at 25°C. Substrates tested were cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and sinapic acid (Sigma-Aldrich). For the competitive assays, Ni-purified enzyme was incubated with 100 μM of each substrate, caffeic acid, coumaric acid and ferulic acid, at 25°C for 10, 20 or 30 minutes (two replicates each). Assays were stopped by adding 100 μL methanol and placing on ice. Products were detected by liquid chromatography-mass spectrometry (LC-MS). The identity of coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA was confirmed by comparing mass and retention time with authentic standards. For competitive assays, peak area of extracted ion chromatograms was used to calculate percentage of products formed. 15 2.3.4 Transient expression of CcAAEs in Nicotiana benthamiana For transient expression in N. benthamiana, the coding sequence of the eight CcAAEs were separately cloned into the pCAMBiA2300U vector. After sequence verification, pCAMBiA vectors carrying the CcAAEs as well as pCAMBiA vectors carrying the previously described genes for myricetin biosynthesis CcFLS, CcCYP2 and CcMYB4 (Irmisch, Ruebsam, et al. 2019) and the genes for MbA biosynthesis CcUGT1, CcUGT2, CcAT, CcUGT3, CcUGT4 and CcUGT5 (all nine genes are referred to as montbretin biosynthetic genes = MBGs) (Irmisch et al. 2018; 2020; Irmisch, Jancsik, et al. 2019) and the pBIN:p19 were individually transformed into Agrobacterium tumefaciens strain C58pMP90. Agrobacterium cultures were prepared as previously described (Irmisch et al. 2020). Briefly, overnight cultures were grown (220 rpm, 28 °C) and used to inoculate 10 mL LB-media containing 50 µg/mL kanamycin, 25 µg/mL rifampicin and 25 µg/mL gentamicin for overnight growth. The following day the cultures were centrifuged (4,000 x g, 5 min) and cells were re-suspended in infiltration buffer (10 mM MES, 10 mM MgCl2, 100 µM acetosyringone, pH 5.6) to a final OD600 of 0.5. After shaking for 2 h at 21°C, combinations were prepared for leaf infiltration by mixing equal volumes of transformed A. tumefaciens. MBGs were used as a control: A. tumefaciens 35Spro:(CcMYB4 + CcFLS + CcCYP2 + CcUGT1 + CcUGT2 + CcUGT3 + CcUGT4 + CcUGT5 + CcAT) + A. tumefaciens pBIN:p19. AAE were individually tested in the background of MBGs: MBGs + A. tumefaciens 35Spro:(CcAAE) (AAE being 1/11 of the mixture; Appendix A.6). The leaves of four-week-old N. benthamiana plants were infiltrated with these Agrobacterium suspensions using a 1-mL needleless syringe to gently push the suspension into the leafâs abaxial surface. Infiltrated leaves were labeled with tape and harvested five days after infiltration. Leaf discs 1 cm in diameter were cut from harvested leaves using a cork borer. Leaf discs were then weighed, and 16 metabolites were extracted using 1 mL of 50% MeOH (v/v) per 100 mg of tissue. Extracts were analyzed using LC-MS. 2.3.5 Reconstruction of flavanone biosynthesis in yeast For heterologous expression in yeast the yeast toolkit for modular cloning (MoClo-YTK) was used (Addgene, MA, USA) (Lee et al. 2015). First, the montbretia flavanone biosynthetic genes, CcCHS2, CcCHI2 (Irmisch, Ruebsam, et al. 2019), and Cc4CL1, Cc4CL2 (this work), were individually cloned into the entry vector, sequenced, paired with a promotor and terminator (Figure 2.6, Appendix A.9) and cloned into a transcriptional unit. Transcriptional units were then combined to multigene constructs containing CcCHS2, CcCHI2 and one of the 4CLs; Cc4CL1 or Cc4CL2 (Appendix A.9). This yielded two flavanone biosynthesis expression modules, each differing in their 4CL. Details about promotors, terminators and parts for the vector backbone can be found in Appendix A.9. Each module was individually transformed into the yeast strain BY4741 (GE Life Sciences, http://www.gelifesciences.com/). For flavanone biosynthesis, a single yeast colony was used to inoculate a starting culture in 5 mL selective uracil dropout media (SD-URA), which was grown overnight at 28 °C and 180 rpm. The following day, 250 µL SD-URA media were inoculated to an OD of 0.1, fed with coumaric or caffeic acid (100 µM end concentration; 2.5 μL of 100 mM stock dissolved in ethanol) and grown in 48-well plates covered with a sterile-breathable sealing film (VWR, ON, Canada) for 67h at 28 °C and 180 rpm. To prevent evaporation, plates were placed in a plastic container under high humidity. Three replicates were grown for each sample and the experiment was repeated two times independently. For metabolite analysis, 50 µL sample was mixed with 50 µL methanol and the supernatant was analysed as described in the LC-MS section. 17 2.3.6 LC-MS analyses LC analysis was done on an Agilent 1100 HPLC (Agilent Technologies, Waldbronn, Germany) with Agilent ZORBAX SB-C18 column (50 x 4.6 mm, 1.8 μm particle size) (Merck, Darmstadt, 370 Germany). In vitro enzyme assays were analyzed using 10 mM aqueous ammonium acetate (Mobile phase A) and acetonitrile plus formic acid (0.2% v/v; Mobile phase B) with the following elution profile: 0 - 5 minutes, 5 â 35 % B in A; 5 to 9 minutes, 35 - 90% B in A. The flow rate was 0.8 mL/min at a column temperature of 50 °C. N. benthamiana extracts and yeast assays were analyzed using aqueous formic acid (0.2% v/v; Mobile phase A) and acetonitrile plus formic acid (0.2% v/v; Mobile phase B). The elution profile for N. benthamiana leaf extracts was: 0 - 0.5 min, 95% A; 0.5 - 5 min, 5 - 20% B in A; 5 - 7 min 90% B in A and 7.1 - 10 min 95% A. The flow rate was 0.8 mL/min at a column temperature of 50 °C. The elution profile for flavanones produced in yeast was: 0 - 0.5 min, 95% A; 0.5 - 5 min, 5 - 40% B in A; 5 - 7 min 90% B in A and 7.1 - 10 min 95% A. The flow rate was 0.8 mL/min at a column temperature of 50 °C. LC was coupled to an Agilent MSD Trap XCT-Plus mass spectrometer using electrospray ionization functioning in negative mode (capillary voltage, 4000 eV; temp, 350 °C; nebulizing gas, 60 psi; dry gas 12 L/min). MS/MS was used to monitor daughter ion formation. Agilent LC/MSD Trap Software 5.2 (Bruker Daltonik) was used for data acquisition and analysis. MbA, MbB, and MbC production in N. benthamiana was quantified using an external MbA standard curve and flavanone production in yeast was quantified using naringenin or eriodictyol standard curves. Compounds were identified using their retention times, molecular masses, and specific fragmentation patterns and by use of authentic standards if they were 18 available (eriodictyol and naringenin, Sigma-Aldrich; MbA and MbB, Tarling et al. 2008; Irmisch et al. 2018). 2.3.7 Statistical analysis One-way analysis of variance (ANOVA) was used to test for significant differences in the accumulation of MbA, MbB and MbC in N. benthamiana. Welchâs two-sample t-test was used to test for differences in flavanone production in yeast. These analyses were conducted in base R 4.0.2 (R Core Team, 2020). 2.3.8 Accession Numbers Nucleotide sequences were deposited in GenBank with the accession numbers MZ944850 (Cc4CL1), MZ944849 (Cc4CL2). Accession numbers or sequences for all other genes/proteins used in this work are indicated in Appendices A.1 and A.2. Previously published transcriptome libraries as well as time course data described in the manuscript are available in the NCBI/GenBank Sequence Read Archive (SRA) under the project PRJNA389589 (SRP108844). 2.4 Results 2.4.1 Identification of AAEs expressed in montbretia corms In order to identify AAEs potentially involved in MbA biosynthesis, we performed a BLASTP search using a set of published AAE sequences from other plant species (Appendix A.1) against a previously described montbretia transcriptome database covering a time course of corm development (Irmisch et al. 2020). We focused on AAEs with high similarity to 4CLs and on AAEs clustering in clades for which enzyme activity with hydroxycinnamic acids has been 19 shown in other species (Gaid et al. 2012; Klempien et al. 2012; S. Lee et al. 2012; Martin et al. 2016; Schnabel et al. 2019; Srividya et al. 2020). We identified 16 contigs encoding full length montbretia AAEs in clades IV, V, VI, and VII (Figure 2.2) (Appendix A.2). Figure 2.1 Phylogeny of montbretia acyl activating enzymes (AAEs). Predicted amino acid sequences of montbretia AAEs that showed expression patterns that matched those of known MbA biosynthetic genes during young corm development were aligned with selected AAEs from other plant species. A neighbour-joining tree was built and tested with 1000 bootstrap replicates. Bootstrap values are displayed at branch nodes. Enzyme clades I, IV, V, VI, VII are labelled [11]. Montbretia enzymes characterized in this work are in bold. Only AAE clades reported to contain enzymes that activate aromatic acids were considered. AtLACS1 was used as the outgroup. Cc: Crocosmia x crocosmiiflora, Gm: Glycine max, At: Arabidopsis thaliana, Pn: Piper nigrum, Os: Oryza sativa, Pv: Panicum virgatum, Sb: Scutellaria baicalensis. [binary] 8898100AtAAE7CcAAE13100CcAAE1599AtBZO1HcCNL99100AtAAE13CcAAE169955AtLACS1VVIIVIIV class 1IVclass 220 All 16 sequences contained the AMP-binding/adenylate-forming (SSGTTXXPKGV, PROSITE PS00455) motif characteristic for AAEs (Shockey and Browse 2011). A phylogeny of the predicted amino acid sequences revealed two contigs that clustered with known 4CLs (Figure 2.2). Cc4CL1 (CcAAE2) was most similar to the monocot subclade of class II 4CLs, thought to be involved in phenylpropanoid metabolism other than lignin biosynthesis; while Cc4CL2 (CcAAE10) clustered with monocot class I 4CLs, involved in monolignol biosynthesis (Cukovic et al. 2001; Y. Li et al. 2015; Ehlting et al. 1999). Cc4CL1 and Cc4CL2 share 60% amino acid identity and 79.7% amino acid similarity. Eleven montbretia contigs clustered with clade V, two with clade VI, and one with clade VII (Figure 2.2). Both clade V and VI AAEs have been shown to contain peroxisomal acyl-CoA ligases, and amino acid sequence analysis revealed that, except for Cc4CL1, Cc4CL2, CcAAE4, CcAAE5, and CcAAE9, all other candidate AAE possess a C-terminal peroxisomal targeting signal (PTS1) (Appendix A.2) (Reumann 2004). To further narrow down the list of montbretia candidate AAEs, temporal patterns of expression in developing corms were compared to previously characterized MbA biosynthetic genes (MBGs). Transcript abundance in counts-per-million for five developmental time points (Irmisch et al. 2020) was used to generate a heatmap and to cluster genes based on expression correlation (Figure 2.3, Appendices A.3, A.4). We had previously shown that MBGs share a common pattern of transcript expression with highest transcript abundance in the early summer in developing young corms (Figure 2.3). Based on their expression clustering with MBGs, eight candidate genes CcAAE1, CcAAE3, CcAAE4, CcAAE5, CcAAE7, CcAAE9, and Cc4CL1 (AAE2) and Cc4CL2 (AAE10) were selected for further characterization. 21 Figure 2.2 Temporal expression profile of montbretia AAEs and previously characterized MbA biosynthetic genes. Heatmap showing relative transcript abundance of AAEs in montbretia young corms at six different time points of corm development. Each value represents the mean counts per million of two biological replicates centered and scaled by transcript. Genes previously characterized as being involved in MbA biosynthesis are shown in gray. Candidate AAEs selected for characterization in this work are bold. Sampling dates in 2016 are indicated at the bottom. 2.4.2 Cc4CL1 and Cc4CL2 activate hydroxycinnamic acids The cDNAs covering the full-length open reading frames of the eight AAEs were cloned using cDNA template made from young corm transcripts, and AAEs were individually expressed in E. coli for functional characterization. For initial tests for 4CL activity, whole protein extracts June 10June 27July 22Aug 16Sept 12Oct 06CcAAE11CcAAE12CcAAE8CcAAE16CcAAE14CcAAE13CcAAE15CcAAE1CcAAE7CcUGT4CcUGT3CcAT1CcFLSCcAAE2 (Cc4CL1)CcUGT1CcUGT5CcAAE3CcAT2CcAAE4CcUGT2CcCYP2CcF3H2CcAAE9CcCYP1CcAAE5CcAAE10 (Cc4CL2)CcAAE6CcF3H1-1.5-1-0.500.511.522 were prepared and assayed with ATP, MgCl2, coenzyme A, and individual phenylpropanoid substrates, specifically cinnamic acid, coumaric acid, caffeic acid, ferulic acid, and sinapic acid. A whole protein extract prepared from E. coli harbouring an empty vector was used as a negative control. Products were identified by liquid chromatography-mass spectrometry (LC-MS). We observed CoA ligase activity with the above-mentioned substrates only for Cc4CL1 and Cc4CL2 (Figure 2.4; Appendix A.5). Whole protein extracts as well as Ni-purified Cc4CL1 and Cc4CL2 converted coumaric acid, caffeic acid, and ferulic acid to the corresponding CoA thioesters coumaroyl-CoA (peak 1; m/z 912), caffeoyl-CoA (peak 2; m/z 928), and feruloyl-CoA (peak 3; m/z 942) (Figure 2.4A). In addition, Cc4CL2 also converted cinnamic acid to cinnamoyl-CoA (m/z 896). No activity was detected with sinapic acid. None of the other tested AAEs showed product formation with any of the tested hydroxycinnamic acids. Protein expression was verified by Western blot (Appendix A.5B). 23 Figure 2.3 Enzyme activities of Cc4CL1 and Cc4CL2. (A) LC-MS product profiles. Enzymes were heterologously expressed in E. coli and Ni-purified protein was assayed with ATP, MgCl2, CoA, and one of the hydroxycinnamic acids, cinnamic acid, coumaric acid, caffeic acid, or ferulic acid. As controls, assays were performed with purified protein extracts of E. coli transformed with the empty vector. Products were analyzed using LC-MS and extracted ion chromatograms (EIC) are shown. Peak 1, tentatively identified as cinnamoyl-CoA (m/z 896); peak 2, coumaroyl-CoA (m/z 912); peak 3, caffeoyl-CoA (m/z 928); peak 4, feruloyl-CoA (m/z 942). n.d. not detected. (B) Relative product formation in competition assays. Cc4CL1 or Cc4CL2 were assayed with a combined set of the three hydroxycinnamic acid substrates, coumaric acid, caffeic acid and ferulic acid, in one reaction. The 100% activity value was defined by the substrate that yielded the most abundant product in the competition assays. Both caffeoyl-CoA and coumaroyl-CoA are required for MbA biosynthesis. In addition, utilization of coumaroyl-, caffeoyl- and feruloyl-CoA by CcAT leads to the formation of MbB, MbA and MbC, respectively. Since Cc4CL1 and Cc4CL2 were both active with caffeic acid and coumaric acid, as well as other hydroxycinnamic acids as substrates, we measured their activity with different substrates in vitro with competition assays. Assays were performed with Ni-Substrate Cc4CL1 Cc4CL2p-Coumaric acid 100.0 ± 21.1% 100.0 ± 1.4 %Caffeic acid 4.2 ± 0.7% 11.4 ± 0.2 %Ferulic acid n.d. 26.0 ± 1.8 %BRelativeintensity (EIC x 1,000,000)10200.10.30.51.53 4 3 4 3 4Retention time (min)5 5 5EIC 912Cc4CL2Cc4CL1ControlStandardEIC 928 EIC 942222333444A55150.10.3121510.51.5510101EIC 896155 50.3150.24 5 6Not availableSubstrate cinnamic acidSubstrate coumaric acidSubstrate caffeic acidSubstrate ferulic acid0.150.124 purified enzyme using equimolar concentrations of the three substrates caffeic acid, coumaric acid and ferulic acid. In competition assays, coumaric acid was the preferred substrate for both Cc4CL1 and Cc4CL2 (Figure 2.4B). Cc4CL1 displayed 4.2 ± 0.7% activity with caffeic acid and no detectable activity with ferulic acid relative to the activity with coumaric acid (set 100%), while Cc4CL2 displayed 11.4 ± 0.2% activity with caffeic acid and 26.0 ± 1.8% with ferulic acid relative to activity with coumaric acid (100%). We observed a lack of stability of the Ni-purified enzyme which prevented us from assessing enzyme kinetic parameters. 2.4.3 CcAAEs did not affect MbA levels when transiently expressed in N. benthamiana We previously established a transient N. benthamiana montbretin pathway expression system in which the expression of MBGs enables MbB and trace amounts of MbA formation (Irmisch et al. 2020). Here, we tested the effect of co-expressing CcAAEs on montbretin levels. We individually co-expressed each of the eight candidate CcAAEs, regardless of their in vitro 4CL activity, with the MBGs (35Spro:(CcMYB4 + CcFLS + CcCYP2 (CYP75B138) + CcUGT1 + CcUGT2 + CcUGT3 + CcUGT4 + CcUGT5 + CcAT) + pBIN:p19) (Appendix A.6) in N. benthamiana leaves. Expression of Cc4CL1 or Cc4CL2 in combination with MBGs did not affect montbretin product profiles relative to expression of MBGs alone (Figure 2.5, Appendix A.7 and A.8). MbB was the major product formed (371.4 ± 31.3 µg/g), followed by MbC (79.9 ± 10.6 µg/g), and lower amounts of MbA (6.3 ± 1.4 µg/g). Beyond Cc4CL1 and Cc4CL2, expression of the other CcAAEs also had no effect on the levels of MbA (F8,18 = 1.566, p = 0.204), MbB (F8,18 = 1.426, p = 0.252) or MbC (F8,18 = 1.064, p = 0.429) (Appendix A.7 and A.8). 25 Figure 2.4 Montbretin accumulation in N. benthamiana co-expressing MbA biosynthetic genes and Cc4CL1 or Cc4CL2. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and MbA biosynthetic genes (= MBGs, [6]) as well as 4CL1 or 4CL2 (see Supporting information S6). Leaves were collected 5 days post-infiltration. Metabolites were extracted with 50% MeOH, analyzed by LC-MS. Data represents mean ± SE (n = 4). MbA (F8,18 = 1.57, p = 0.204), MbB (F8,18 = 1.43, p = 0.25), MbC (F8,18 = 1.06, p = 0.43), n.s. not significant 2.4.4 Utilizing Cc4CLs to reconstruct flavanone biosynthesis in Saccharomyces cerevisiae Unlike plants, S. cerevisiae does not have endogenous 4CL activity which is essential for flavonoid biosynthesis. We tested the utility of Cc4CL1 and Cc4CL2 in S. cerevisiae to convert exogenously fed p-coumaric acid to p-coumaroyl-CoA, and exogenously fed caffeic acid to caffeoyl-CoA, to support formation of the flavanones naringenin and eriodictyol (Figure 2.6). Naringenin and eriodictyol are precursors for myricetin, which is the core building block of MbA (Figure 2.1). For metabolic engineering of montbretin in yeast, 4CL activity would also enable the formation of p-coumaroyl-CoA or caffeoyl-CoA as acyl donors for CcAT in MbB or MbA biosynthesis, respectively. MBG MBG + Cc4CL1 MBG + Cc4CL2 0 0.1 0.2 0.3 0.4 MbA MbB MbC mg/g (FW) 26 Figure 2.5 Reconstruction of flavanone biosynthesis in Saccharomyces cerevisiae using montbretia 4CLs. (A) Flavanone biosynthesis module. For expression in S. cerevisiae, expression modules containing montbretia chalcone synthase (CcCHS2), chalcone isomerase (CcCHI2) and one of two 4-coumaroyl CoA ligases (4CLs), montbretia Cc4CL1 or Cc4CL2, were constructed. (B) Schematic representation of flavanone biosynthetic pathway. Coumaric acid or caffeic acid are converted into naringenin or eriodictyol, respectively. Lactone formation occurs as a side reaction. (C,D) Engineering flavanone production in S. cerevisiae. Flavanone biosynthesis modules (see A) differing in their Cc4CL genes were heterologously expressed in S. cerevisiae. Genes were constitutively expressed from the same low copy plasmid. Yeast cultures were grown for 67h in 48-well plates, extracted and analysed for flavanone formation using LC-MS. Representative extracted ion chromatograms (EIC) (C) and amounts of produced naringenin or eriodictyol are shown (n = 2) (D). Peak 1, tentatively identified as p-coumaroyltriacetic acid lactone; peak 2, naringenin; peak 3, tentatively identified as caffeoyltriacetic acid lactone; peak 4, eriodictyol. Changes in flavanone levels were tested using Welchâs two-sample t-tests (naringenin: p = 0.27; eriodictyol: p < 0.001). We expressed Cc4CL1 or Cc4CL2 together with the previously described CcCHS2 and CcCHI2 in S. cerevisiae (Irmisch, Ruebsam, et al. 2019). Constructs were assembled using the yeast toolkit for modular cloning (MoClo-YTK) (M. E. Lee et al. 2015) to express all three genes 51526104.5 5.5 6.5 7.5Caffeic acid fedCoumaric acid fedEIC 271EIC 287EIC 271EIC 287123 4Retention time (min)Relative intensity(EIC x 100 000)HTB2CcCHS2ENO1 RPL18BCcCHI2SSA1 HHF1Cc4CLTDH1ABCD naringenin eriodictyolCcCHS2CcCHI2Cc4CL1CcCHS2CcCHI2Cc4CL2135mg * L-1p < 0.00127 from one plasmid using constitutive promotors of different strengths (Figure 2.6A, Appendix A.9). Plasmids were transformed into S. cerevisiae BY4741, and cultures were fed with p-coumaric acid or caffeic acid and grown in 48-well plates for 67 h. Naringenin or eriodictyol formation was detected using LC-MS. Both 4CLs activated p-coumaric or caffeic acid to provide hydroxycinnamoyl-CoA thioesters necessary for flavanone production as shown by the formation of naringenin or eriodictyol, respectively (Figure 2.6C, D). Hydroxycoumaroyl triacetic lactone formation occurred as a side product (Appendix A.10). Expression of Cc4CL1 resulted in slightly higher levels of naringenin production (5.8 mg/L) when p-coumaric acid fed, compared to Cc4CL2 (5.2 mg/L); eriodictyol production (caffeic acid fed) was higher with the expression of Cc4CL2 (2.3 mg/L) compared to Cc4CL1 (1.5 mg/L). Naringenin titers were about 3-fold higher compared to eriodictyol titers. The formation of the side product, triacetic acid lactone was more pronounced when caffeic acid was fed and eriodictyol produced. No product formation could be detected using a GFP control vector or when no hydroxycinnamic acid was fed. 2.5 Discussion Previous research on the biosynthesis of the anti-diabetic and anti-obesity compound MbA has mainly focused on the unique assembly of MbA from a suite of common plant metabolites (i.e., MbA building blocks), specifically the flavonoid myricetin, the phenylpropanoid caffeoyl-CoA, and the UDP-sugars UDP-rhamnose, UDP-glucose and UDP-xylose (Irmisch et al. 2018; 2020; Irmisch, Ruebsam, et al. 2019; Irmisch, Jancsik, et al. 2019). The central phenylpropanoid pathway (Vogt 2010) produces the precursor coumaroyl-CoA for myricetin, and it also produces caffeoyl-CoA, thus feeding twice into the MbA biosynthetic system. While caffeoyl-CoA is required for the acylation reaction in the MbA assembly pathway, 28 the similar phenylpropanoids coumaroyl-CoA and feruloyl CoA provide the acyl functionalities for MbB and MbC, respectively (Figure 2.1). MbB and MbC have no significant potential for treatment of diabetes or obesity (Tarling et al. 2008), but their biosynthesis in montbretia or in metabolically engineered tobacco or yeast competes with MbA production (Tarling et al. 2008; Irmisch, Ruebsam, et al. 2019). Here, we investigated montbretia AAEs with a focus on 4CLs and their ability to convert caffeic acid, coumaric acid or ferulic acid into caffeoyl-CoA, coumaroyl-CoA or feruloyl-CoA, respectively. We identified two Cc4CLs, both of which showed activity with all three hydroxycinnamic acids and an apparent preference for coumaric acid as substrate relative to caffeic acid and ferulic acid. While expression of AAEs failed to enhance MbA biosynthesis in tobacco, we demonstrated that both Cc4CL1 and Cc4CL2 can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in metabolically engineered yeast. The CcATs that catalyze the formation of montbretins can use caffeoyl-CoA, coumaroyl-CoA and feruloyl-CoA, thus leading to the formation of MbA, MbB and MbC (Irmisch et al. 2018). Given the lack of CcAT substrate specificity it is possible that preferential biosynthesis of any one of these three montbretins might be controlled by differences in the availability of caffeoyl-CoA, coumaroyl-CoA or feruloyl-CoA. In addition, all three montbretins require coumaroyl-CoA for the myricetin core. We hypothesized that montbretia corms might express a set of different Cc4CLs with distinct substrate specificities as a means to regulate the formation of caffeoyl-CoA, coumaroyl-CoA and feruloyl-CoA. However, the results of this work indicate that this is not the case. Substrate promiscuity of 4CLs has also been observed in other plant species (Lindermayr et al. 2002; Gui, Shen, and Li 2011; Sun et al. 2013; Lin et al. 2015; Liu et 29 al. 2018). The majority of 4CLs have been studied in dicot species where they generally display a preference for p-coumaric acid or caffeic acid (Lindermayr et al. 2002; Costa et al. 2005; H. C. Chen et al. 2013). Fewer 4CLs have been characterized in monocots. In rice (Oryza sativa), switchgrass (Panicum virgatum), and great millet (Sorghum bicolor), 4CLs generally prefer coumaric acid and ferulic acid relative to caffeic acid (Gui, Shen, and Li 2011; Xu et al. 2011; Saballos et al. 2012). This pattern holds true for Cc4CL2, which displayed preference for p-coumaric acid and ferulic acid compared to caffeic acid, while Cc4CL1 did not accept ferulic acid in competition assays. In addition to bona fide 4CLs, some AAEs outside the 4CL clade have been reported to use phenylpropanoid substrates. For example, AAEs in clades V, VI, and VII contribute to benzenoid metabolism through activation of cinnamic acid (Gaid et al. 2012; Klempien et al. 2012; Block et al. 2014; Soubeyrand et al. 2019). In yew (Taxus media) AAEs outside of the 4CL clade can activate hydroxycinnamic acids with TmAAE12 preferring caffeic acid in vitro (Srividya et al. 2020). Arabidopsis (Arabidopsis thaliana) clade V AAEs also activate p-coumaric acid and/or caffeic acid (Block et al. 2014; Kienow et al. 2008). In contrast, none of the CcAAEs outside of the 4CL clade showed activity with hydroxycinnamic acids. Thus, 4CL activity in montbretia appears to be limited to the bona fide 4CL clade, and does not contribute to selective biosynthesis of MbA relative to other montbretins. 4CLs cluster within clade IV of the AAE family. Within clade IV, two functionally distinct sub-clades exist, class I and class II. Enzymes involved in lignin biosynthesis cluster within class I, while class II contains 4CLs that are likely involved in soluble phenylpropanoid metabolism, including flavonoid biosynthesis (Cukovic et al. 2001; Y. Li et al. 2015; Ehlting et 30 al. 1999). Cc4CL1 clusters within class II 4CLs, accepts coumaric acid and caffeic acid as substrates, and its gene expression pattern matches that of MBGs during young corm development. Cc4CL2 clusters within class I, accepts coumaric acid, caffeic acid as well as ferulic acid as substrates, but its expression profile pattern does not match that of other MBGs. Both Cc4CL1 and Cc4CL2 show high transcript abundance in early summer. These observations suggest that Cc4CL1 is more likely to contribute to MbA biosynthesis while Cc4CL2 may be involved in lignin biosynthesis. We previously engineered N. benthamiana for the production of MbA by transiently co-expressing a suite of genes for myricetin and MbA biosynthesis (Irmisch et al. 2020; Irmisch, Ruebsam, et al. 2019). This system yielded small amounts of less than 10 µg MbA per g fresh weight of tobacco leaf material, while levels of MbB and MbC were about 60-fold and 15-fold, respectively, higher. We showed here that co-expression of AAEs, including Cc4CL1 and Cc4CL2, did not alter these product profiles suggesting that 4CL activity is not rate limiting for overall montbretin production in N. benthamiana. However, future work shall focus on the possibility to enhance caffeoyl-CoA formation in N. benthamiana in order to increase the relative ratio and overall yield of MbA. This may be achieved through selection or mutagenesis of a 4CL enzyme with higher substrate preference for caffeic acid, as is observed with At4CL2 (Costa et al. 2005). N. benthamiana and yeast each have unique advantages and also provide challenges for metabolic engineering of MbA production. In contrast to N. benthamiana, which naturally produces all of the MbA building blocks, MbA biosynthesis in yeast will require more extensive engineering for most of the MbA building blocks, in addition to engineering of the MbA 31 assembly pathway. However, the fact that yeast does not produce caffeic acid/caffeoyl-CoA and coumaric acid/coumaroyl-CoA, may allow for better control of MbA versus MbB production in yeast by controlled feeding of caffeic acid. CHS which is the entry enzyme for flavonol biosynthesis efficiently uses caffeic acid although coumaric acid is generally the preferred substrate (Schröder 2000). Here we demonstrated the feasibility of producing the flavanones eriodictyol and naringenin, two possible precursors to myricetin, from caffeic acid and coumaric acid, respectively, by heterologous co-expression of Cc4CLs with montbretia flavanone biosynthetic genes. The yields of these flavanones were comparable to eriodictyol and naringenin production reported for yeast ectopically expressing flavanone biosynthetic genes, while higher levels can be achieved through genomic integration and pathway optimization (Jiang, Wood, and Morgan 2005; Chemler, Yan, and Koffas 2006; Lyu et al. 2017; Lehka et al. 2017). In future work we will further optimize eriodictyol production from caffeic acid in yeast, similar to the previously described naringenin production (Lyu et al. 2017; Lehka et al. 2017). 32 Chapter 3: Improving montbretin A yields in engineered Nicotiana benthamiana 3.1 Summary The plant kingdom has always been a useful source of therapeutic molecules for humans. Montbretin A (MbA) is a complex acylated flavonol glycoside found in the underground storage organs of montbretia (Crocosmia crocosmiiflora). MbA was discovered as a highly potent and specific human pancreatic α-amylase inhibitor, making it an attractive antidiabetic treatment candidate. Since Crocosmia is not adapted for large scale cultivation, and MbAâs complex structure renders chemical synthesis infeasible, we are developing Nicotiana benthamiana as a host for heterologous MbA production. Previously acquired knowledge of the MbA biosynthesis pathway has enabled the production of trace amounts of MbA in N. benthamiana. This system produced much higher quantities of inactive side-products, differing from MbA in the identity of their acyl groups. MbB possesses a coumaroyl group, and MbC a feruloyl group. We identified genes involved in the biosynthesis of the acyl precursor caffeoyl-CoA. We investigated the early steps in caffeoyl-CoA biosynthesis. Two montbretia enzymes, CcHCT3 and CcCSE2 both caused 5-10x fold increases in MbA concentrations when expressed in Nicotiana. In addition, a chlorogenic acid esterase from Ustilago maydis was expressed in N. benthamiana which depleted chlorogenic acid stores in the plant. When co-expressed with montbretin biosynthetic genes, this esterase resulted in a 5-fold increase in MbA levels relative to MBGs. Using untargeted metabolomics, we investigated how these genes improved MbA biosynthesis and influenced phenolic metabolism. 33 3.2 Introduction The plant kingdom continues to provide a variety of phytochemicals useful for human health. Montbretin A (MbA) is a potent and specific human pancreatic α-amylase inhibitor discovered in the ornamental plant montbretia (Tarling et al. 2008). MbAâs unique activity makes it an attractive candidate for the treatment of diabetes and obesity. Its ability to influence post-prandial blood glucose levels have been demonstrated in animal trials (Yuen et al. 2016). MbA is a complex acylated flavonol glycoside, defined as myricetin 3-O-(glucosyl-6â-O-caffeoyl)-1,2-Î²-D-glucosyl 1,2-α-L-rhamnoside 4â-O-Î±-L-rhamnosyl 1,4-β-D-xyloside (Asada, Hirayama, and Furuya 1988; Williams et al. 2015). In the event of MbA being approved for clinical use, chemical synthesis, or agricultural production of montbretia are not considered to be able to meet expected demand. An alternative method for producing rare and complex plant-sourced molecules is through metabolic engineering of a heterologous host. This approach requires in-depth knowledge of MbA biosynthesis. In this context, our lab has elucidated the MbA biosynthetic pathway. MbA is sequentially assembled by five UDP-dependent glycosyltransferases and one BAHD-acyltransferase (CcAT) (Irmisch et al. 2018; Irmisch, Jancsik, et al. 2019; Irmisch et al. 2020). The third step in MbA biosynthesis is the acylation of myricetin glycosyl rhamnoside (MRG) into mini-MbA catalyzed by CcAT. CcAT is promiscuous regarding acyl donors and can thus accept at least both coumaroyl-CoA and caffeoyl-CoA (Irmisch et al. 2018). The incorporation of coumaroyl results in the production of montbretin B (MbB) and the presence of a feruloyl group results in montbretin C (MbC), both of which are weak HPA inhibitors (Williams et al. 2015). While montbretia produces MbA as a major product, smaller amounts of MbB and MbC are also produced. 34 Transient expression of montbretin biosynthetic genes (MBGs) in N. benthamiana resulted in the accumulation of small amounts of MbA (<10 µg/g FW; Irmisch et al. 2020). However, MbB is produced in much larger amounts, likely due to higher availability of coumaroyl-CoA in N. benthamiana. This indicates that increased levels of MbA could be achieved by redirecting biosynthesis away from MbB and towards MbA in N. benthamiana. We originally hypothesized that montbretia corms preferentially produce MbA by preferentially producing caffeoyl CoA relative to coumaroyl CoA based on substrate preference of montbretia 4-coumaroyl-CoA ligase (Cc4CL) activity. We tested this hypothesis by characterization of two different Cc4CLs and their expression in N. benthamiana (Chapter 2). However, co-expression of montbretia 4CLs with MBGs in N. benthamiana did not have an impact on MbA or MbB levels, and both 4CL enzymes were found to use coumaric acid as well as caffeic acid as substrates (Sunstrum et al., in press). With the goal of improving MbA yields by increasing caffeoyl-CoA supply to CcAT, we are exploring other enzymes of the phenylpropanoid pathway involved in the conversion of coumaroyl-CoA to caffeoyl-CoA. We hypothesize that co-expression of one or more of these enzymes with MBGs may modify precursor supply to favor MbA production. The phenylpropanoid pathway is ubiquitous in land plants, being the source of most phenolic compounds produced by plants (Figure 3.1). The pathway diverges from aromatic amino acid biosynthesis through the deamination of phenylalanine by phenylalanine ammonia lyase (PAL; Barros et al. 2016). The resulting cinnamic acid is then oxidized by a cinnamyol-4-hydroxylase (C4H) which produces p-coumaric acid. The following hydroxylation of p-coumaric acid to produce caffeic acid is now considered to occur at the level of the shikimate ester of p-coumaric acid (Schoch et al. 2001; Takeda et al. 2018). The p-coumaric acid is activated by a 35 4CL and ligated to shikimate by the action of a hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT). The 3â position of coumaroyl shikimate is then hydroxylated by coumarate-3-hydroxylase (C3H) in the CYP98 family (Alber et al. 2019; Schoch et al. 2001; Nair et al. 2002). C3H may act as a heterodimer with C4H (Chen et al. 2011). Caffeoyl shikimate is then either hydrolyzed by a caffeoylshikimate esterase (CSE) yielding caffeic acid (requiring activation by a 4CL) and shikimate or is directly converted to caffeoyl-CoA through the reverse action of an HCT. It was suggested that HCT catalyzed a reverse reaction to produce caffeoyl-CoA directly (Hoffmann et al. 2003) and this activity has been demonstrated in vitro (Kreighauser et al. 2021). Some recent studies suggest that CSE is required to produce caffeoyl-CoA in vivo (Vanholme et al. 2013; de Vries et al. 2021; Ha et al. 2016). However, some monocots such as maize (Zea mays) and switchgrass (Brachypodium virgatum) lack CSE homologs in their genomes, suggesting that there is not a universally conserved path to caffeoyl-CoA in plants. 36 Figure 3.1 General phenylpropanoid pathway. Adapted from Vanholme et al., 2013. Another strategy for metabolic engineering would be exploiting compounds abundantly produced by the host. Chlorogenic acid (CGA; caffeoyl quinate) is an abundant phenolic metabolite in N. benthamiana. CGA is biosynthesized from caffeoyl-CoA and quinic acid by enzymes closely related to HCTs. We hypothesize that this large pool of bound caffeic acid could be released from CGA and channeled towards MbA production. Ustilago maydis, a pathogenic fungi, expresses a CGA esterase (UmChlE; Nieter et al., 2015) that could be used to cleave CGA and release caffeic acid. This caffeic acid could then be activated by a native or transiently expressed 4CL and be used for MbA production. 37 Here we will test if overexpressing montbretia HCT, C3H, and CSE in N. benthamiana can be used to alter metabolic flux of the phenylpropanoid metabolism towards increased levels of caffeoyl-CoA, which may then become accessible for metabolic engineering of MbA production in N. benthamiana. In addition, UmChlE presents an opportunity to improve caffeoyl-CoA supply by cleaving the large CGA pool in N. benthamiana. Co-expression of these genes, either individually or together, could contribute to increasing the availability of caffeoyl-CoA to CcAT, thus increasing MbA yields. This chapter describes the expression of phenylpropanoid biosynthetic genes from montbretia, and UmChlE with the goal of increasing MbA levels in transformed N. benthamiana. For the genes that caused an improved in MbA yields, we carried out untargeted metabolomics to assess the impact they had on the broader metabolic landscape of transformed N. benthamiana and to inform future metabolic engineering efforts. 3.3 Materials and Methods 3.3.1 Cloning A BLASTP search was conducted on the previously established montbretia corm transcriptome database using characterized HCT, CSE, and C3H genes as query sequences. + Chlorogenic acid Quinic acid Caffeic acid MbA Figure 3.2 Intended reaction for UmChlE expression in N. benthamiana. 38 Resulting montbretia phenylpropanoid pathway enzyme coding sequences were amplified from previously produced young corm cDNA (Irmisch et al. 2018), and UmChlE was amplified from Ustilago maydis gDNA provided by Dr. Matthias Kretschmer (Kronstad lab). Polymerase chain reaction (PCR) products for one CcC4H, three CcC3Hs, three CcCSEs, three CcHCTs, and UmChlE were separated by agarose gel electrophoresis and gel-extracted using the Monarch® DNA Gel Extraction Kit (NEB) according to manufacturerâs instruction. Resulting fragments were ligated into the pJET1.2/blunt vector (Thermo Fisher Scientific) and used to transform NEB 5-α E. coli strain. Transformed E. coli was cultured in 3.5 ml LB broth at 37°C overnight, and plasmids were purified using the Monarch® Plasmid Miniprep Kit (NEB) and sequenced using Sanger sequencing. Confirmed ORFs were then sub-cloned into a pCAMBIA2300U vector with USER-cloning (Geu-Flores et al., 2007). Open reading frames were amplified from pJet clones with Q5U DNA polymerase (NEB), with primers containing a single uracil which is used to generate 3â overhangs when excised. Sequences are found in Appendix B.1. 3.3.2 Transient expression in N. benthamiana pCAMBIA2300 vectors carrying genes required for myricetin biosynthesis CcFLS, CcCYP2, and CcMYB4 (Irmisch, Ruebsam, et al. 2019), and the genes for MbA biosynthesis CcUGT1, CcUGT2, CcAT, CcUGT3, CcUGT4, and CcUGT5 (Irmisch et al. 2018; 2020; Irmisch, Jancsik, et al. 2019), and the pBIN:p19 vector were provided by Dr. Sandra Irmisch. pCambia constructs were individually transformed into A. tumefaciens strain C58pMP90. 200 µg plasmid DNA was added to chemically competent Agrobacterium cells. After freezing in liquid N2 and thawing at 37°C, cells were incubated on ice for 30 minutes then 1 mL LB was added. After shaking for 2h at 28°C, cells were resuspended in 100 µL LB. Cells were spread on LB agar plates containing kanamycin (kan; 50 µg/mL), rifampicin (rif; 25 µg/mL), and gentamycin (gent; 39 25 µg/mL) and incubated for two days at 28°C. Resulting colonies or previously established glycerol stocks were used to inoculate a 5 mL overnight culture (220 rpm, 28 °C) in LB (kan 50 µg/mL, rif 25 µg/mL, gent 25 µg/mL). 1 mL from this culture was used to inoculate a final 10 mL overnight culture (kan 50 µg/mL, rif 25 µg/mL, gent 25 µg/mL). Cells were centrifuged at 4,300 x g and resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, 100 µM acetosyringone, pH 5.6) to a final OD600 of 0.5. After shaking for 2 h at 21°C, combinations were prepared for leaf infiltration by mixing equal volumes of transformed A. tumefaciens. To screen for effects on montbretin biosynthesis, genes required for mini-MbA biosynthesis were used as a control: A. tumefaciens 35Spro:(CcMYB4 + CcFLS + CcCYP2 + CcUGT1 + CcUGT2 + CcAT) + A. tumefaciens pBin:p19, as the complete MbA assembly pathway was not elucidated at the time. Candidate caffeoyl-CoA biosynthetic genes were individually tested in the background of mini-MBGs (Appendix B.2). The leaves of four-week-old N. benthamiana plants were infiltrated with these Agrobacterium suspensions using a 1-mL needleless syringe to gently push the suspension into the leafâs abaxial surface. Infiltrated leaves were labeled with tape and harvested five days after infiltration. Leaf discs 1 cm in diameter were cut from harvested leaves using a cork borer. Leaf discs were then weighed, and metabolites were extracted using 1 mL of 50% MeOH (v/v) per 100 mg of tissue. Extracts were analyzed using LC-MS. Mini-montbretins and montbretins were quantified using an external MbA standard curve. For the substrate feeding experiment, four-week-old N. benthamiana plants were infiltrated with Agrobacterium strains harbouring mini-MBGs, mMBGs + A. tumefaciens 35Spro:(Cc4CL1), or mMBGs + A. tumefaciens 35Spro:(Cc4CL2). Four days after infiltration, leaf discs 1 cm in diameter were cut from harvested leaves using a cork borer and soaked in one of four solutions: (1) water, (2) 200 µM CoA, (3) 1 µM caffeic acid, or (4) 200 µM CoA and 1 µM 40 caffeic acid together. Since caffeic acid was originally dissolved in DMSO, 0.1% (v/v) DMSO was added to each solution not containing caffeic acid. The following day (5 days post-infiltration), leaf discs were extracted as described above and extracts were analyzed by LC-MS. 3.3.3 LC-MS analyses N. benthamiana extracts were analyzed for montbretin levels as described in section 2.2.6. Metabolic profiling for intermediate identification and metabolomics was conducted on an Agilent 1290 Infinity UHPLC (Agilent Technologies GmbH, Waldbronn, Germany) with Waters Atlantis T3 column (2.1 x 100 mm, 5 µm particle size) using aqueous formic acid (0.2% v/v; mobile phase A) and acetonitrile plus formic acid (0.2% v/v; mobile phase B). The elution profile was 0-10 min, 92% A; 10-25 min, 8-20% B in A; 25-37 min, 20-80% B in A; 37-38 min, 97% A. The flow rate was 0.4 mL/min at a column temperature of 30°C. This LC was coupled to an Agilent 6530 Accurate Mass Q-TOF mass spectrometer equipped with an electrospray ion source operated in negative ionization mode (capillary voltage, 3500 eV; temp, 325°C; nebulizing gas, 30 psi; dry gas 12 L/min). MassHunter workstation software (version B.08.00, 2016) was used for data acquisition and processing. Quantification was based on an external MbA standard curve. 3.3.4 Untargeted metabolomics analyses Accurate mass data for samples expressing MBGs, MBGs + CcHCT3, MBGs + CcCSE2, or MBGs + UmChlE were further analyzed to carry out an untargeted metabolomics analysis. Raw data was centroided and converted to .mzML format using proteowizard MSConvert v3.0.2 (Chambers et al. 2012). Parameters for xcms peak picking, retention time correction, and 41 grouping were optimized with two samples expressing MBGs using the âIPOâ package 1.16.0 (Libiseller et al. 2015) implemented in R 3.60 (Appendix B.3). All converted files were uploaded to the xcms online platform (xcmsonline.scripps.edu/) (Gowda et al. 2014) and analyzed using optimized parameters. Peak area normalization, scaling, and univariate and multivariate statistical analyses were conducted using the MUMA 1.4 R package (Gaude et al. 2013) implemented in R 4.0.3. Peak areas were scaled using Pareto scaling. For the univariate analysis, either Welchâs T test or Mann-Whitneyâs U test was used depending on the result of Shapiro-Wilks test for normality. The Benjamini-Hochberg procedure was used to correct p-values for multiple testing at α = 0.05. 3.4 Results 3.4.1 Effects of phenylpropanoid biosynthetic genes on montbretin levels in engineered N. benthamiana In previous research we achieved accumulation of small amounts of MbA (<10 µg/g FW) and its precursor mini-MbA (105 ± 11 µg/g FW) by transient expression of montbretia genes in N. benthamiana (Irmisch et al. 2020; Irmisch, Ruebsam, et al. 2019). In both cases, mini-MbA and MbA were both surpassed by substantially higher levels of mini-MbB (1448 ± 69 µg/g FW) and MbC (124 ± 16 µg/g FW), which possess a coumaroyl moiety in the place of caffeoyl. In order to improve MbA yields by increasing the availability of caffeoyl-CoA, we identified a set of genes encoding putative phenylpropanoid genes in montbretia (Figure 3.1). Three HCT and CSE sequences and one C3H sequence were identified and co-expressed with mini-MbA biosynthetic genes (mini-MBGs; (35Spro:(CcMYB4 + CcFLS + CcCYP2 + CcUGT1 + CcUGT2 + CcAT) + pBIN:p19)), as the MbA assembly pathway was not yet fully elucidated at the time. When mini-MBGs were expressed in N. benthamiana, mini-MbB (1448 ± 69 µg/g FW) was the 42 dominant product, produced in ~14x and ~12 times higher amounts than mini-MbA (105 ± 11 µg/g FW) and mini-MbC (124 ± 16 µg/g FW), respectively. The co-expression of CcHCT3 or CcCSE2 with mini-MBGs both resulted in a substantial increase in mini-MbA yields, accompanied by decreased mini-MbB and increased mini-MbC levels (Figure 3.2). CcHCT3 caused the greatest increase in mini-MbA levels, reaching 434 ± 60 µg/g (FW), followed by CcCSE2 reaching 410 ± 136 µg/g (FW). The expression of CcHCT1, CcHCT2, CcCSE1, CcCSE3, or CcC3H1 did not result in altered mini-montbretin levels. Figure 3.3 Screening phenylpropanoid biosynthetic genes for improvements in mini-MbA yields in Nicotiana benthamiana. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and mini-MbA biosynthetic genes (=mMBGs) as well as a set of montbretia phenylpropanoid biosynthetic genes (Appendix B.3). Leaves were collected 5 days post-infiltration. Metabolites were extracted with 50% MeOH and analyzed by LC-MS. Data represent mean ± SE (n = 3). Changes were identified using ANOVA (F7,16 = 7.1, p < 0.001) followed by Tukeyâs post-hoc test. 0200400600mMBGs mMBGs +CcHCT1mMBGs +CcHCT2mMBGs +CcHCT3mMBGs +CcCSE1mMBGs +CcCSE2mMBGs +CcCSE3mMBGs +CcC3H-1µg/g mini-MbA (FW)a aaa aab b43 3.4.2 Confirmation of HCT3 and CSE2 activities with the complete MbA pathway Once the complete MbA biosynthetic pathway was elucidated, the effects of CcHCT3 and CcCSE2 on MbA levels were confirmed by co-expressing them with the MbA biosynthetic genes (MBGs; 35Spro:(CcMYB4 + CcFLS + CcCYP2 + CcUGT1 + CcUGT2 + CcUGT3 + CcUGT4 + CcUGT5 + CcAT) + pBIN:p19) in N. benthamiana. When MBGs were expressed, MbA levels reached 7.7 ± 1.8 µg/g FW. Consistent with their impact on mini-MbA levels, both genes caused an increase in MbA levels when co-expressed with MBGs. CcHCT3 and CcCSE2 caused MbA levels to reach 83.7 ± 3.3 and 43.5 ± 5.2 µg/g FW, respectively (Figure 3.3). Expression of CcHCT3 resulted in a decrease in MbB levels from 289.8 ± 24.1 µg/g FW to 166.6 ± 25.7 µg/g FW, and increased MbC levels from 66.7 ± 8.0 µg/g FW to 141.8 ± 20.7 µg/g FW. Expression of CcCSE resulted in a decrease in MbB levels from 289.8 ± 24.1 µg/g FW to 166.6 ± 25.7 µg/g FW, and increased MbC levels from 66.7 ± 8.0 µg/g FW to 141.8 ± 20.7 µg/g FW. Figure 3.4 Montbretin accumulation in N. benthamiana co-expressing mini-MbA biosynthetic genes and various phenylpropanoid biosynthetic genes. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and MbA biosynthetic genes (=MBGs, Irmisch et al., 2020), CcHCT3, and CcCSE2 (Appendix B.3). Leaves were collected 5 0100200300MBGS MBGS+CcHCT3MBGs+CcCSE2µg/g (FW) MbAMbBMbCaccbbb bbc44 days post-infiltration. Metabolites were extracted with 50% MeOH and analyzed by LC-MS. Data represent mean ± SE (n = 4). MbA (F3,12 = 12.1, p = <0.001), MbB (F3,12 = 10.1, p < 0.01), MbC (F3,12 = 5.3, p < 0.05). 3.4.3 Ustilago maydis chlorogenic acid esterase CGA is an abundant phenolic metabolite in Nicotiana species and many other plants (Niggeweg, Michael, and Martin 2004). CGA is caffeoyl-quinate, indicating that N. benthamiana naturally produces large amounts of caffeoyl-CoA which is used for CGA production. U. maydis, a pathogenic fungus, produces a CGA esterase (UmChlE), which cleaves CGA and releases caffeic acid and quinic acid (Nieter et al. 2015). We hypothesized that expressing this enzyme in N. benthamiana could improve MbA production by releasing caffeic acid to be activated and incorporated into MbA. UmChlE was cloned from U. maydis genomic DNA and co-expressed with MBGs in N. benthamiana (Figure 3.4). UmChlE expression depleted the CGA pool in transformed leaves. In addition, this effect was accompanied by a strong increase in free caffeic acid and quinic acid (Figure 3.7). Co-expression of UmChlE with MBGs increased MbA levels 5-fold to 38.3 ± 7.3 µg/g FW, decreased MbB levels 2-fold to 143.0 ± 20.1 µg/g FW, and increased MbC levels 2-fold to 137.6 ± 27.1 µg/g FW. Intended reaction for UmChlE expression in N. benthamiana. 45 Figure 3.5 Montbretin accumulation in N. benthamiana co-expressing MbA biosynthetic genes and a chlorogenic acid esterase from Ustilago maydis (UmChlE). N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and MbA biosynthetic genes (=MBGs, Irmisch et al., 2020) and UmChlE (Appendix B.3). Leaves were collected 5 days post-infiltration. Metabolites were extracted with 50% MeOH and analyzed by LC-MS. Data represent mean ± SE (n = 4). MbA (F1,6 = 16.9, p < 0.01), MbB (F1,6 = 21.92, p < 0.01), MbC (F1,6 = 6.3, p < 0.05). 3.4.4 Substrate feeding Our results indicate that caffeoyl-CoA is limiting MbA biosynthesis in N. benthamiana leaves expressing MBGs. Research described in Chapter 2 found that co-expressing a 4CL with MBGs did not improve MbA levels, suggesting that 4CL substrates, caffeic acid and coenzyme A, may be limiting for MbA production in N. benthamiana. We conducted a substrate feeding experiment with both caffeic acid and Coenzyme A to transformed N. benthamiana expressing mini-MBGs together with a previously identified 4CL (Figure 3.5). Leaf discs were taken from infiltrated leaves expressing mini-MBGs, mini-MBGs + Cc4CL1, or mini-MBGs + Cc4CL2 four days post infiltration and soaked in water, 1 mM caffeic acid, 200 µM CoA, or caffeic acid and CoA together. Leaf discs were extracted in 50% MeOH (v/v) the following day and extracts were MBGs+UmChlE0100200300MBGsµg/g (FW)MbAMbBMbCabccd d46 analyzed by LC-MS. In accordance with previous results, the co-expression of Cc4CL1 or Cc4CL2 did not affect mini-montbretin amounts in N. benthamiana. Additionally, feeding caffeic acid did not significantly impact mini-montbretin yields. Feeding coenzyme A was associated to an increase in mini-MbA levels (Figure 3.5). Figure 3.6 Substrate feeding experiment in N. benthamiana co-expressing mini-MbA biosynthetic genes (MBGs) and both montbretia 4CLs. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and mini-MbA biosynthetic genes (=mMBGs), Cc4CL1, and Cc4CL2 (Appendix B.3). Four days after infiltration, leaf discs 1 cm in diameter were cut from harvested leaves using a cork borer and soaked in one of four solutions: (1) Water, (2) 200 µM CoA, (3) 1 µM caffeic acid, or (4) 200 µM CoA and 1 µM caffeic acid together.The following day (5 days post-infiltration), leaf discs were extracted as described above and extracts were analyzed by LC-MS. Data represent mean ± SE (n = 2). 3.4.5 Metabolomics The co-expression of CcHCT3, CcCSE2, and UmChlE with MBGs in N. benthamiana resulted in ~11, ~6, and ~5-fold increases, respectively, in MbA levels relative to the expression of MBGs alone. This analysis targeted specifically montbretins, and further metabolic CoA WaterCaffeic acidWatermMBGs mMBGs+Cc4CL1mMBGs+Cc4CL2mMBGs mMBGs+Cc4CL1mMBGs+Cc4CL20500100005001000µg/g (FW)Mini-MbAMini-MbBMini-MbC47 characterization of these plants is required to lay the foundation for future engineering and shed light on the mechanisms behind the observed increases in MbA yields. We conducted an untargeted metabolomic analysis of extracts of leaves expressing MBGs alone, or MBGs with either CcHCT3, CcCSE2, or UmChlE using HPLC-Quadrupole Time-of-Flight MS. Resulting raw data was analyzed using xcms (xcmsonline.scripps.edu/) for peak detection and alignment. The xcms analysis detected 4312 metabolite peaks throughout the 16 analyzed samples. Principal component analysis (PCA) was used to assess overall metabolic differences between sample groups (Figure 3.6). Principal components 1 and 2 accounted for 63.9% and 9.9% of variance, respectively. This analysis shows samples expressing UmChlE as a distinct cluster, while samples expressing MBGs, and MBGs + CcHCT3 or MBGs + CcCSE2 are more similar to each other with minimal separation between each group. Figure 3.7 Metabolic changes induced by expression of CcCSE2, CcHCT3, and UmChlE. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-MBGs MBGs + CcCSE2 MBGs + CcHCT3 MBGs + UmChlE 125 (2.9%) 2 (0.0%) 29 (0.7%) 128 (3.0%) 48 (1.1%) 34 (0.8%) 16 (0.4%) 89 (2.1%) 35 (0.8%) 51 (1.2%) 616 (14.3%) 145 (3.4%) 95 (2.2%) 120 (2.8%) 2780 (64.5%) MBGs MBGs + CcCSE2 MBGs + CcHCT3 MBGs + UmChlE -0.25 0.00 0.25 0.50 -0.25 0.00 0.25 PC1 (67.72%) PC2 (8.41%) A B 48 promoter-gene constructs for myricetin and MbA biosynthetic genes (=MBGs, Irmisch et al., 2020), CcCSE2, CcHCT3, or UmChlE. Leaf extracts were analyzed by HPLC-QTOF. Peaks were identified using xcms and differential peaks were identified with the âmumaâ R package. A: Distribution of peaks throughout treatment groups. B: Principal component analysis based on abundance of m/z peaks. Peak areas were scaled using pareto scaling. Pairwise comparisons were made comparing samples expressing MBGs as a control to MBGs + CcHCT3, MBGs + CcCSE2, and MBGs + UmChlE. Co-expression of CcCSE2, CcHCT3, and UmChlE with MBGs induced significant changes in 71, 2, and 506 metabolites, respectively. These differential peaks were categorized based on potential relevance to MbA biosynthesis. The accurate mass for each differential peak was entered in the Metlin mass spectrometry database (metlin.scripps.edu/). A total of 272 peaks were flagged as âpotentially of interestâ if at least one database result was a flavonoid or flavonoid derivative, or a phenylpropanoid pathway intermediate. The identities of a set of montbretin pathway intermediates and precursors (Figure 3.7) were confirmed using enzymatic products (caffeoyl shikimate, coumaroyl shikimate, coumaroyl quinate) or authentic standards (quinic acid, shikimic acid, coumaric acid, caffeic acid, and chlorogenic acid). Each of these compounds except for coumaroyl-shikimate and free shikimic acid were identified in transformed N. benthamiana leaf extracts. The expression of CcHCT3 increased caffeoyl-shikimate levels ~16-fold relative to other treatment groups (Figure 3.7). Caffeoyl-shikimate was also decreased by ~35% and 37% with the expression of CcCSE2 and UmChlE, respectively, compared to the expression of MBGs. As expected, chlorogenic acid levels were nearly depleted when UmChlE was expressed. This effect was accompanied by 24 and 8-fold increases in caffeic acid and quinic acid, respectively. UmChlE also caused a depletion of coumaroyl-quinate accompanied by a ~2-fold increase in coumaric acid, likely due 49 to substrate promiscuity of this enzyme. Peaks corresponding to coumaroyl-CoA or caffeoyl-CoA were not identified. Figure 3.8 Abundance of intermediates in caffeoyl-CoA biosynthesis in N. benthamiana plants expressing MBGs and genes improving MbA yields. N. benthamiana leaves were infiltrated with different combinations of A. tumefaciens transformed with plasmids carrying the 35S-promoter-gene constructs for myricetin and MbA biosynthetic genes (=MBGs, Irmisch et al., 2020), CcCSE2, CcHCT3, or UmChlE. 5 days post-infiltration, leaf discs were extracted as described above and extracts were analyzed by HPLC-QTOF. Peaks were detected with xcms and identified using enzymatic products (Caffeoyl-shikimate, coumaroyl-shikimate, coumaroyl-quinate) or authentic standards (caffeic acid, coumaric acid, chlorogenic acid, shikimic acid, quinic acid). Coumaric acid and Relative intensity (EIC x 10 000)Caffeic acid (m/z 179.0350)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE0200400600a a abCaffeoyl-shikimate (m/z 335.0767)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE051015a a abCoumaric acid (m/z 163.0399)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE01234 n.s.Chlorogenic acid (m/z 353.0875)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE050010001500 aa abCoumaroyl-quinate (m/z 337.0930)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE050100150200abbcQuinic acid (m/z 191.0563)MBGs MBGs + CcCSE2MBGs + CcHCT3MBGs + UmChlE0100200300a a ab50 shikimic acid were not identified in the samples. Data represent mean ± SE (n = 4). Statistical differences were determined using ANOVA followed by Tukeyâs post-hoc test. n.s. = not-significant. 3.5 Discussion This chapter describes steps towards optimizing supply of the MbA precursor caffeoyl-CoA with the objective to improve heterologous MbA production in N. benthamiana. Two montbretia genes, CcHCT3 and CcCSE2, were found to increase MbA yields 11 and 6-fold, respectively. In addition, the co-expression of a chlorogenic acid esterase from the pathogenic fungus U. maydis (UmChlE) with MBGs was found to improve MbA yields 5-fold. A substrate feeding experiment was conducted in an attempt to identify biosynthetic bottlenecks in caffeoyl-CoA supply. Finally, an untargeted metabolomic dataset was generated as a core resource to inform future metabolic engineering efforts. The discovery of the MbA assembly pathway (Irmisch, Jancsik, et al. 2019; Irmisch et al. 2018; 2020) and montbretia myricetin biosynthetic genes (Irmisch, Ruebsam, et al. 2019) enabled the production of trace amounts of MbA in N. benthamiana. This was accompanied by considerable production of MbB and MbC, indicating that substantial yield gains could be achieved by altering the acyl group. By co-expressing genes involved in the conversion of coumaroyl-CoA to caffeoyl-CoA with MBGs, the resulting increase in caffeoyl-CoA pools should result in increased MbA yields. CcHCT3 and CcCSE2 both improved MbA yields, while CcC3H1 did not, suggesting that the bottleneck in caffeoyl-CoA biosynthesis is not at the meta-hydroxylation step. CcHCT3 and CcCSE2 catalyze two different steps in caffeoyl-CoA biosynthesis, yet have similar effects on montbretin levels when co-expressed with MBGs. The identification of phenylpropanoid pathway intermediates and substrates in transformed N. benthamiana leaves can provide insights 51 on the mechanism of action of CcHCT3, CcCSE2, and UmChlE. HCT enzymes initiate the conversion of coumaroyl-CoA to caffeoyl-CoA by ligating coumaroyl to shikimic acid (Kreighauser et al. 2021). The expression of CcHCT3 induced a 16-fold increase in its downstream product caffeoyl-shikimate (Figure 3.7). This is likely due to efficient hydroxylation of coumaroyl-shikimate to caffeoyl-shikimate by an endogenous N. benthamiana C3H. The increase in caffeoyl-shikimate levels supported the identity of CcHCT3 as a bona fide HCT that is increasing MbA levels by initiating the conversion of coumaroyl-CoA to caffeoyl-CoA. The expected mechanism by which CcCSE2 is improving MbA yields is by cleaving caffeoyl-shikimate and releasing caffeic acid, which is then activated by a 4CL to produce caffeoyl-CoA. When co-expressed with MBGs, CcCSE2 caused a 35% decrease in caffeoyl-shikimate and a 15% increase in caffeic acid, both of which were not statistically significant. However, these changes follow the expected trends and support this hypothesis of CcCSE2âs mechanism of action. The expression of UmChlE had the intended effect of cleaving the large pool of CGA in N. benthamiana to release caffeic acid and quinic acid (Nieter et al. 2015; Figure 3.4). It also resulted in a 5-fold increase in MbA levels accompanied by decreased MbB levels and increased MbC levels. Increased MbA levels are likely due to increased caffeic acid being converted to caffeoyl-CoA by an endogenous N. benthamiana 4CL. This increased caffeoyl-CoA would then be used for MbA production. The fact that co-expression of a montbretia 4CL with UmChlE (Appendix B.3) did not impact mini-MbA levels may be due to sub-cellular localization and warrants further investigation. In addition, the decrease in MbB levels caused by the co-expression of UmChlE with MBGs was unexpected. The same effect observed with CcHCT3 and CcCSE2 could have been attributed to decreased coumaroyl-CoA availability which was 52 converted to caffeoyl-CoA. However, the release of caffeic acid caused by UmChlE which is converted to caffeoyl-CoA should not come at the expense of coumaroyl-CoA. We can then speculate that the observed decrease in MbB levels may be due to increased incorporation of caffeoyl-CoA into a limited montbretin pool. Our results show that caffeoyl-CoA is limiting for MbA production in N. benthamiana. Caffeoyl-CoA can be produced from caffeic acid and coenzyme A by a 4CL enzyme. It is possible that one of these two precursors is limiting. Thus, we conducted a substrate feeding experiment feeding caffeic acid and/or coenzyme A to transformed N. benthamiana leaves expressing mini-MBGs. If either or both precursors is limiting for caffeoyl-CoA supply, we would expect to see an increase in mini-MbA levels. Feeding Coenzyme A resulted in a statistically significant increase in mini-MbA levels (Figure 3.5), independent of caffeic acid feeding. Based on this result, engineering upstream Coenzyme A biosynthesis may be beneficial to MbA production in N. benthamiana. However, this result should be replicated due to small sample size. This follow-up experiment should consider timing of feeding and other methods of substrate feeding such as infiltration (Lau and Sattely 2015). All analyses described to date have been limited to observing the effect of overexpressing certain genes on montbretin and phenylpropanoid pathway intermediate levels. An untargeted metabolomic analysis was carried out to lay the foundation for future metabolic engineering. The 4304 unknown peaks identified in transformed N. benthamiana leaves likely contain many metabolites that would be useful for MbA engineering. These could include undesirable side-products or products from pathways competing for precursors. Further research is required to identify these compounds using a combination of database searches, and MS/MS analyses using machine learning algorithms such as SIRIUS 4.0 (Dührkop et al. 2019). The apparent reliability 53 of the xcms pipeline with the targeted analysis described above provides support for the larger metabolomic dataset as a resource for future metabolic engineering of MbA production in N. benthamiana. 54 Chapter 4: Conclusion The characterization of MbAâs biosynthetic pathway was a critical step in establishing a heterologous MbA production system (Irmisch et al. 2020; Irmisch, Jancsik, et al. 2019; Irmisch et al. 2018). The remaining obstacle consists of assuring adequate supply of MbAâs building blocks. In N. benthamiana, engineering myricetin supply from kaempferol was achieved by expressing a set of montbretia genes involved in myricetin biosynthesis (Irmisch, Ruebsam, et al. 2019). As the required UDP-sugars and hydroxycinnamoyl-CoA precursors are naturally produced in N. benthamiana, this previous work enabled heterologous MbA production. The remaining obstacle relates to the relative availabilities of coumaroyl-CoA and caffeoyl-CoA. Coumaroyl-CoA is required for myricetin biosynthesis, and caffeoyl-CoA is required for the acylation step of MbA assembly. Originally, the major product in engineered N. benthamiana was MbB, indicating that coumaroyl-CoA was being preferentially incorporated at both steps. However, MbA is the predominant product in montbretia corms (Irmisch et al. 2018). Since coumaroyl-CoA and caffeoyl-CoA are required in a 1:1 ratio in MbA biosynthesis, it is unclear how montbretia regulates the selective incorporation of coumaroyl-CoA and caffeoyl-CoA at their respective biosynthetic steps. Since MbB is the predominant product in engineered N. benthamiana, this thesis aimed to increase the availability of caffeoyl-CoA for the acylation step, thus improving MbA yields. We identified a set of montbretia genes involved in the biosynthesis of the MbA precursors coumaroyl-CoA and caffeoyl-CoA. The 4CL-like gene family was studied in the most detail. We hypothesized that montbretia may regulate MbA production by selectively activating coumaric acid or caffeic acid. Two 4CLs were identified, both having broad substrate specificities. Based on temporal expression pattern and phylogeny, Cc4CL1 is likely involved in 55 MbA biosynthesis, whereas Cc4CL2 is likely involved in lignin biosynthesis. The co-expression of either Cc4CL1 or Cc4CL2 with MBGs did not result in improved MbA yields in N. benthamiana. The search was thus expanded to genes in the phenylpropanoid pathway involved in the conversion of coumaroyl-CoA to caffeoyl-CoA. A series of montbretia genes were individually co-expressed with MBGs. Two enzymes, CcHCT3 and CcCSE2, were found to improve MbA levels 11- and 6-fold, respectively. Their effect on the levels of key pathway intermediates supported their identities as HCT and CSE enzymes. In addition, we hydrolyzed the large pool of chlorogenic acid produced by N. benthamiana by expressing a chlorogenic acid esterase from Ustilago maydis. This resulted in the accumulation of large amounts of caffeic acid, and in a 5-fold increase in MbA levels. To lay the groundwork for further engineering in this system, we conducted an untargeted metabolomic analysis of transformed N. benthamiana leaves. This work provides some insights into how montbretia preferentially produces MbA, but a conclusive understanding requires additional research. We ruled out the possibility of selective activation of caffeic acid, since the two Cc4CLs expressed in montbretia corms are not selective toward this substrate and it is likely that only one of these two 4CLs is involved in MbA biosynthesis in montbretia. One possibility is that intermediates are channeled in metabolons where coumaroyl-CoA and caffeoyl-CoA are incorporated at their respective steps. Such structures have been described both in lignin and soluble phenylpropanoid biosynthesis (Gou et al. 2018; Nakayama, Takahashi, and Waki 2019; Obata 2019). Another possibility is that the need for coumaroyl-CoA is bypassed by using caffeoyl-CoA as a substrate for myricetin biosynthesis. CcCHS2, which catalyzes this reaction has been shown to accept caffeoyl-CoA as a substrate when expressed in yeast for flavanone production (Figure 2.6). Even if this is not the 56 pathway used in vivo by montbretia, CcCHS2 could be engineered for the preferred use of caffeoyl-CoA as a metabolic engineering strategy. The co-expression of CcHCT3 with MBGs in N. benthamiana resulted in 83.7 ± 3.3 µg MbA /g FW, the highest yield achieved yet. However, further yield improvements will be required to provide a truly efficient MbA production system. This work mainly focused on shifting biosynthesis in engineered N. benthamiana from MbB towards MbA, but further yield gains could be achieved via other approaches. Certain experiments described above should be repeated, refined, and expanded upon. First, the experiments testing the co-expression of multiple phenylpropanoid biosynthetic genes should be repeated using the full MbA pathway, with more biological replicates to account for inter-sample variability, and with all combinations tested in the same experiment. Second, the metabolomic dataset should be further analyzed. This work limited compound identification to a small set of key phenylpropanoid pathway intermediates, and a large set of metabolites of interest were left unidentified. Additional analysis including MS/MS combined with machine learning algorithms for structural elucidation should reveal useful leads for metabolic engineering. Also, a transcriptomic dataset of transformed N. benthamiana leaves could be analyzed alongside the already established metabolome to correlate transcript and metabolite abundance. Observing the effect of expressing phenylpropanoid biosynthetic genes such as CcHCT3 and CcCSE2 may reveal insights in the regulation of phenylpropanoid biosynthesis. This transcriptomic dataset would enable the use of gene downregulation for metabolic engineering. The production of MbC in N. benthamiana is caused either by the incorporation of feruloyl-CoA by CcAT, or by the activity of a non-specific O-methyltransferase. Further work could focus on downregulating CCoAMT, the o-methyltransferase responsible for the conversion of caffeoyl-57 CoA to feruloyl-CoA (Bureau et al. 2007). A transcriptome analysis of transformed N. benthamiana would also shed light on the expression levels of transiently expressed montbretin pathways genes. This would determine whether each transgene is expressed at similar levels. Any identified discrepancies could be corrected by adjusting promoter strength (Engler et al. 2014). Another concern which may impact MbA yields is the use of individual plasmids for each co-expressed gene in N. benthamiana. While this approach was advantageous for rapid screening of various gene combinations, expressing the full MbA pathway with CcHCT3 involves the expression of 10 genes on individual plasmids, and it is possible that a substantial proportion of N. benthamiana cells do not receive the full set of 10 genes. The development of multi-gene constructs would solve this problem. MbA is now one of many plant specialized metabolites that can be produced in transformed N. benthamiana at a lab scale (Gülck et al. 2020; van Herpen et al. 2010; Reed and Osbourn 2018; Cankar et al. 2015; Martí et al. 2020; Davis et al. 2020). While engineered yeast is used commercially for metabolite production, N. benthamiana is not currently employed for this application. However, N. benthamiana is used for the production of therapeutic proteins and vaccines (Leblanc, Waterhouse, and Bally 2021; Goulet et al. 2019). Considering the numerous opportunities remaining to increase MbA yields in this system, achieving industrial MbA production in N. benthamiana is feasible. 58 Bibliography Alber, Annette V., Hugues Renault, Alexandra Basilio-Lopes, Jean Etienne Bassard, Zhenhua Liu, Pascaline Ullmann, Agnès Lesot, et al. 2019. âEvolution of Coumaroyl Conjugate 3-Hydroxylases in Land Plants: Lignin Biosynthesis and Defense.â Plant Journal, 924â36. https://doi.org/10.1111/tpj.14373. Asada, Yoshihisa, Yutaka Hirayama, and Tsutomu Furuya. 1988. âAcylated Flavonols from Crocosmia crocosmiiflora.â Phytochemistry 27 (5): 1497â1501. https://doi.org/10.1016/0031-9422(88)80223-1. Barros, Jaime, Juan C. Serrani-Yarce, Fang Chen, David Baxter, Barney J. Venables, and Richard A. Dixon. 2016. âRole of Bifunctional Ammonia-Lyase in Grass Cell Wall Biosynthesis.â Nature Plants 2 (6): 1â9. https://doi.org/10.1038/NPLANTS.2016.50. Block, Anna, Joshua R. Widhalm, Abdelhak Fatihi, Rebecca E. Cahoon, Yashitola Wamboldt, Christian Elowsky, Sally A. Mackenzie, et al. 2014. âThe Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis.â Plant Cell 26 (5): 1938â48. https://doi.org/10.1105/tpc.114.125807. Bureau, T., Kevin C. Lam, Ragai K. Ibrahim, Bahareh Behdad, and Selvadurai Dayanandan. 2007. âStructure, Function, and Evolution of Plant O-Methyltransferases.â Genome 50 (11): 1001â13. https://doi.org/10.1139/G07-077. Cankar, Katarina, Esmer Jongedijk, Martin Klompmaker, Timotej Majdic, Roland Mumm, Harro Bouwmeester, Dirk Bosch, and Jules Beekwilder. 2015. â(+)-Valencene Production in Nicotiana benthamiana Is Increased by down-Regulation of Competing Pathways.â Biotechnology Journal 10 (1): 180â89. https://doi.org/10.1002/biot.201400288. Chambers, Matthew C., Brendan MacLean, Robert Burke, Dario Amodei, Daniel L. Ruderman, 59 Steffen Neumann, Laurent Gatto, et al. 2012. âA Cross-Platform Toolkit for Mass Spectrometry and Proteomics.â Nature Biotechnology 30 (10): 918â20. https://doi.org/10.1038/nbt.2377. Chemler, Joseph A., Yajun Yan, and Mattheos A.G. Koffas. 2006. âBiosynthesis of Isoprenoids, Polyunsaturated Fatty Acids and Flavonoids in Saccharomyces cerevisiae.â Microbial Cell Factories 5: 1â9. https://doi.org/10.1186/1475-2859-5-20. Chen, Feng, Dorothea Tholl, Jörg Bohlmann, and Eran Pichersky. 2011. âThe Family of Terpene Synthases in Plantsâ: A Mid-Size Family of Genes for Specialized Metabolism That Is Highly Diversified throughout the Kingdomâ 66 (1): 212â29. https://doi.org/10.1111/j.1365-313X.2011.04520.x. Chen, H.-C., Q. Li, C. M. Shuford, J. Liu, D. C. Muddiman, R. R. Sederoff, and V. L. Chiang. 2011. âMembrane Protein Complexes Catalyze Both 4- and 3-Hydroxylation of Cinnamic Acid Derivatives in Monolignol Biosynthesis.â Proceedings of the National Academy of Sciences 108 (52): 21253â58. https://doi.org/10.1073/pnas.1116416109. Chen, Hsi Chuan, Jina Song, Cranos M. Williams, Christopher M. Shuford, Jie Liu, Jack P. Wang, Quanzi Li, et al. 2013. âMonolignol Pathway 4-Coumaric Acid: Coenzyme A Ligases in Populus Trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme a Ligation Fluxes.â Plant Physiology 161 (3): 1501â16. https://doi.org/10.1104/pp.112.210971. Chiasson, J. L., R. G. Josse, J. A. Hunt, C. Palmason, N. W. Rodger, S. A. Ross, E. A. Ryan, M. H. Tan, and T. M.S. Wolever. 1994. âThe Efficacy of Acarbose in the Treatment of Patients with Non-Insulin- Dependent Diabetes Mellitus. A Multicenter Controlled Clinical Trial.â Annals of Internal Medicine. https://doi.org/10.7326/0003-4819-121-12-199412150-00004. 60 Cho, N. H., J. E. Shaw, S. Karuranga, Y. Huang, J. D. da Rocha Fernandes, A. W. Ohlrogge, and B. Malanda. 2018. âIDF Diabetes Atlas: Global Estimates of Diabetes Prevalence for 2017 and Projections for 2045.â Diabetes Research and Clinical Practice. https://doi.org/10.1016/j.diabres.2018.02.023. Costa, Michael A., Diana L. Bedgar, Syed G.A. Moinuddin, Kye Won Kim, Claudia L. Cardenas, Fiona C. Cochrane, Jay M. Shockey, et al. 2005. âCharacterization in Vitro and in Vivo of the Putative Multigene 4-Coumarate:CoA Ligase Network in Arabidopsis: Syringyl Lignin and Sinapate/Sinapyl Alcohol Derivative Formation.â Phytochemistry 66: 2072â91. https://doi.org/10.1016/j.phytochem.2005.06.022. Cragg, Gordon M., Saul A. Schepartz, Matthew Suffness, and Michael R. Grever. 1993. âThe Taxol Supply Crisis. New NCI Policies for Handling the Large-Scale Production of Novel Natural Product Anticancer and Anti-HIV Agents.â Journal of Natural Products 56 (10): 1657â68. https://doi.org/10.1021/np50100a001. Cukovic, Daniela, Jürgen Ehlting, Jessica A. VanZiffle, and Carl J. Douglas. 2001. âStructure and Evolution of 4-Coumarate: Coenzyme A Ligase (4CL) Gene Families.â Biological Chemistry 382 (4): 645â54. https://doi.org/10.1515/BC.2001.076. Davis, Katharine, Danai S. Gkotsi, Duncan R.M. Smith, Rebecca J.M. Goss, Lorenzo Caputi, and Sarah E. OâConnor. 2020. âNicotiana Benthamiana as a Transient Expression Host to Produce Auxin Analogs.â Frontiers in Plant Science 11 (November): 1â9. https://doi.org/10.3389/fpls.2020.581675. Dixon, Richard A., and Dieter Strack. 2003. âPhytochemistry Meets Genome Analysis, and Beyond.........â Phytochemistry. https://doi.org/10.1016/S0031-9422(02)00712-4. Dührkop, Kai, Markus Fleischauer, Marcus Ludwig, Alexander A. Aksenov, Alexey V. Melnik, 61 Marvin Meusel, Pieter C. Dorrestein, Juho Rousu, and Sebastian Böcker. 2019. âSIRIUS 4: A Rapid Tool for Turning Tandem Mass Spectra into Metabolite Structure Information.â Nature Methods 16 (4): 299â302. https://doi.org/10.1038/s41592-019-0344-8. Ehlting, Jürgen, Daniela Büttner, Qing Wang, Carl J. Douglas, Imre E. Somssich, and Erich Kombrink. 1999. âThree 4-Coumarate:Coenzyme A Ligases in Arabidopsis Thaliana Represent Two Evolutionarily Divergent Classes in Angiosperms.â Plant Journal 19 (1): 9â20. https://doi.org/10.1046/j.1365-313X.1999.00491.x. Engels, Benedikt, Pia Dahm, and Stefan Jennewein. 2008. âMetabolic Engineering of Taxadiene Biosynthesis in Yeast as a First Step towards Taxol ( Paclitaxel ) Productionâ 10: 201â6. https://doi.org/10.1016/j.ymben.2008.03.001. Engler, Carola, Mark Youles, Ramona Gruetzner, Tim Martin Ehnert, Stefan Werner, Jonathan D.G. Jones, Nicola J. Patron, and Sylvestre Marillonnet. 2014. âA Golden Gate Modular Cloning Toolbox for Plants.â ACS Synthetic Biology 3 (11): 839â43. https://doi.org/10.1021/sb4001504. Fett-neto, Arthur G, Frank Dicosmo, W E Reynolds, and Ko Sakata. 1992. âCell Culture of Taxus as a Source of the Antineoplastic Drug Taxol.â Nature Biotechnology 10: 1572â75. Gaid, Mariam M., Debabrata Sircar, Andreas Müller, Till Beuerle, Benye Liu, Ludger Ernst, Robert Hänsch, and Ludger Beerhues. 2012. âCinnamate: CoA Ligase Initiates the Biosynthesis of a Benzoate-Derived Xanthone Phytoalexin in Hypericum Calycinum Cell Cultures.â Plant Physiology 160 (3): 1267â80. https://doi.org/10.1104/pp.112.204180. Galanie, Stephanie, Kate Thodey, Isis J. Trenchard, Maria Filsinger Interrante, and Christina D. Smolke. 2015. âComplete Biosynthesis of Opioids in Yeast.â Science 349 (6252): 1095â1100. 62 Gao, Hui, Dong Mei Guo, Wen Juan Liu, Jin Hua Ran, and Xiao Quan Wang. 2012. âEvolution of the 4-Coumarate:Coenzyme A Ligase (4CL) Gene Family: Conserved Evolutionary Pattern and Two New Gene Classes in Gymnosperms.â Journal of Systematics and Evolution 50 (3): 195â205. https://doi.org/10.1111/j.1759-6831.2012.00187.x. García-Pérez, Luis Emilio, María Ãlvarez, Tatiana Dilla, Vicente Gil-Guillén, and Domingo Orozco-Beltrán. 2013. âAdherence to Therapies in Patients with Type 2 Diabetes.â Diabetes Therapy. https://doi.org/10.1007/s13300-013-0034-y. Gaude, Edoardo, Francesca Chignola, Dimitrios Spiliotopoulos, Andrea Spitaleri, Michela Ghitti, Jose M Garcia-Manteiga, Silvia Mari, and Giovanna Musco. 2013. âMuma, An R Package for Metabolomics Univariate and Multivariate Statistical Analysis.â Current Metabolomics 1 (2): 180â89. https://doi.org/10.2174/2213235x11301020005. Gou, Mingyue, Xiuzhi Ran, Dwight W. Martin, and Chang Jun Liu. 2018. âThe Scaffold Proteins of Lignin Biosynthetic Cytochrome P450 Enzymes.â Nature Plants 4 (5): 299â310. https://doi.org/10.1038/s41477-018-0142-9. Goulet, Marie Claire, Linda Gaudreau, Marielle Gagné, Anne Marie Maltais, Ann Catherine Laliberté, Gilbert Ãthier, Nicole Bechtold, et al. 2019. âProduction of Biopharmaceuticals in Nicotiana BenthamianaâAxillary Stem Growth as a Key Determinant of Total Protein Yield.â Frontiers in Plant Science 10 (June). https://doi.org/10.3389/fpls.2019.00735. Gowda, Harsha, Julijana Ivanisevic, Caroline H. Johnson, Michael E. Kurczy, H. Paul Benton, Duane Rinehart, Thomas Nguyen, et al. 2014. âInteractive XCMS Online: Simplifying Advanced Metabolomic Data Processing and Subsequent Statistical Analyses.â Analytical Chemistry 86 (14): 6931â39. https://doi.org/10.1021/ac500734c. Gui, Jinshan, Junhui Shen, and Laigeng Li. 2011. âFunctional Characterization of Evolutionarily 63 Divergent 4-Coumarate: Coenzyme a Ligases in Rice.â Plant Physiology 157 (2): 574â86. https://doi.org/10.1104/pp.111.178301. Gülck, Thies, J. K. Booth, Carvalho, B. Khakimov, C. Crocoll, M. S. Motawia, B. L. Møller, J. Bohlmann, and N. J. Gallage. 2020. âSynthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana Benthamiana and Saccharomyces Cerevisiae.â Journal of Natural Products 83 (10): 2877â93. https://doi.org/10.1021/acs.jnatprod.0c00241. Ha, Chan Man, Luis Escamilla-Trevino, Juan Carlos Serrani Yarce, Hoon Kim, John Ralph, Fang Chen, and Richard A. Dixon. 2016. âAn Essential Role of Caffeoyl Shikimate Esterase in Monolignol Biosynthesis in Medicago Truncatula.â The Plant Journal 86: 363â75. Herpen, Teun W.J.M. van, Katarina Cankar, Marilise Nogueira, Dirk Bosch, Harro J. Bouwmeester, and Jules Beekwilder. 2010. âNicotiana Benthamiana as a Production Platform for Artemisinin Precursors.â PLoS ONE 5 (12). https://doi.org/10.1371/journal.pone.0014222. Hoffmann, Laurent, Stéphane Maury, Françoise Martz, Pierrette Geoffroy, and Michel Legrand. 2003. âPurification, Cloning, and Properties of an Acyltransferase Controlling Shikimate and Quinate Ester Intermediates in Phenylpropanoid Metabolism.â Journal of Biological Chemistry 278 (1): 95â103. https://doi.org/10.1074/jbc.M209362200. Ikram, Nur K.B.K., and Henrik T. Simonsen. 2017. âA Review of Biotechnological Artemisinin Production in Plants.â Frontiers in Plant Science 8 (November): 1â10. https://doi.org/10.3389/fpls.2017.01966. Irmisch, Sandra, Sharon Jancsik, Macaire Man Saint Yuen, Lufiani L. Madilao, and Joerg Bohlmann. 2020. âComplete Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin 64 A.â Plant Physiology 184 (1): 97â109. https://doi.org/10.1104/pp.20.00522. Irmisch, Sandra, Sharon Jancsik, Macaire M.S. Yuen, Lufiani L. Madilao, and Jörg Bohlmann. 2019. âBiosynthesis of the Anti-Diabetic Metabolite Montbretin A: Glucosylation of the Central Intermediate Mini-MbA.â Plant Journal 100 (5): 879â91. https://doi.org/10.1111/tpj.14493. Irmisch, Sandra, Seohyun Jo, Christopher R Roach, Sharon Jancsik, Macaire M.S. Yuen, Lina Madilao, Mark Mark OâNeil-Johnson, Russell Williams, Stephen G Withers, and Joerg Bohlmann. 2018. âDiscovery of UDP-Glycosyltransferases and BAHD-Acyltransferases in the Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin A.â The Plant Cell 30 (August): 1864â86. https://doi.org/10.1105/tpc.18.00406. Irmisch, Sandra, Henriette Ruebsam, Sharon Jancsik, Macaire M.S. Yuen, Lina Madilao, and Joerg Bohlmann. 2019. âFlavonol Biosynthesis Genes and Their Use in a Synthetic Biology Approach to Engineering the Plant Antidiabetic Metabolite Montbretin A.â Plant Physiology 180 (July): 1277â90. https://doi.org/10.1104/pp.19.00254. Jiang, Hanxiao, Karl V. Wood, and John A. Morgan. 2005. âMetabolic Engineering of the Phenylpropanoid Pathway in Saccharomyces Cerevisiae.â Applied and Environmental Microbiology 71 (6): 2962â69. https://doi.org/10.1128/AEM.71.6.2962-2969.2005. Kienow, Lucie, Katja Schneider, Michael Bartsch, Hans Peter Stuible, Hua Weng, Otto Miersch, Claus Wasternack, and Erich Kombrink. 2008. âJasmonates Meet Fatty Acids: Functional Analysis of a New Acyl-Coenzyme A Synthetase Family from Arabidopsis Thaliana.â Journal of Experimental Botany 59 (2): 403â19. https://doi.org/10.1093/jxb/erm325. Klempien, Antje, Yasuhisa Kaminaga, Anthony Qualley, Dinesh A. Nagegowda, Joshua R. Widhalm, Irina Orlova, Ajit Kumar Shasany, et al. 2012. âContribution of CoA Ligases to 65 Benzenoid Biosynthesis in Petunia Flowers.â Plant Cell 24 (5): 2015â30. https://doi.org/10.1105/tpc.112.097519. Kolde, Raivo. 2015. âPheatmapâ: Pretty Heatmaps.â R Package Version 1.0.8. Kreighauser, Lucie, Samuel Knosp, Etienne Greinenberger, Kanade Tatsumi, Desirée D Gütle, Iben Sørensen, Laurence Herrgott, et al. 2021. âFunction of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE Is Evolutionarily Conserved in Embryophytes.â The Plant Cell 33 (5): 1472â91. Kumar, Sudhir, Glen Stecher, and Koichiro Tamura. 2016. âMEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.â Molecular Biology and Evolution 33 (7): 1870â74. https://doi.org/10.1093/molbev/msw054. Lau, Warren, and Elizabeth S. Sattely. 2015. âSix Enzymes from Mayapple That Complete the Biosynthetic Pathway to the Etoposide Aglycone.â Science 349 (6253): 1224â28. Lavhale, Santosh G., Raviraj M. Kalunke, and Ashok P. Giri. 2018. âStructural, Functional and Evolutionary Diversity of 4-Coumarate-CoA Ligase in Plants.â Planta 248 (5): 1063â78. https://doi.org/10.1007/s00425-018-2965-z. Leblanc, Zacharie, Peter Waterhouse, and Julia Bally. 2021. âPlant-Based Vaccines: The Way Ahead?â Viruses 13 (1): 1â13. https://doi.org/10.3390/v13010005. Lee, Michael E., William C. DeLoache, Bernardo Cervantes, and John E. Dueber. 2015. âA Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.â ACS Synthetic Biology 4 (9): 975â86. https://doi.org/10.1021/sb500366v. Lee, Shinyoung, Yasuhisa Kaminaga, Bruce Cooper, Eran Pichersky, Natalia Dudareva, and Clint Chapple. 2012. âBenzoylation and Sinapoylation of Glucosinolate R-Groups in Arabidopsis.â Plant Journal 72 (3): 411â22. https://doi.org/10.1111/j.1365-66 313X.2012.05096.x. Lehka, Beata Joanna, Michael Eichenberger, Walden Emil Bjørn-Yoshimoto, Katherina Garcia Vanegas, Nicolaas Buijs, Niels Bjerg Jensen, Jane Dannow Dyekjær, Håvard Jenssen, Ernesto Simon, and Michael Naesby. 2017. âImproving Heterologous Production of Phenylpropanoids in Saccharomyces Cerevisiae by Tackling an Unwanted Side Reaction of Tsc13, an Endogenous Double-Bond Reductase.â FEMS Yeast Research 17 (1): 1â12. https://doi.org/10.1093/femsyr/fox004. Li, Jianhua, Ishmael Mutanda, Kaibo Wang, Lei Yang, Jiawei Wang, and Yong Wang. 2019. âChloroplastic Metabolic Engineering Coupled with Isoprenoid Pool Enhancement for Committed Taxanes Biosynthesis in Nicotiana Benthamiana.â Nature Communications 10: 1â12. https://doi.org/10.1038/s41467-019-12879-y. Li, Yi, Jeong Im Kim, Leonard Pysh, and Clint Chapple. 2015. âFour Isoforms of Arabidopsis Thaliana 4-Coumarate: CoA Ligase (4CL) Have Overlapping yet Distinct Roles in Phenylpropanoid Metabolism.â Plant Physiology 169 (December): 2409â21. https://doi.org/10.1104/pp.15.00838. Libiseller, Gunnar, Michaela Dvorzak, Ulrike Kleb, Edgar Gander, Tobias Eisenberg, Frank Madeo, Steffen Neumann, et al. 2015. âIPOâ: A Tool for Automated Optimization of XCMS Parameters.â BMC Bioinformatics 16 (118): 1â10. https://doi.org/10.1186/s12859-015-0562-8. Lin, Chien Yuan, Jack P. Wang, Quanzi Li, Hsi Chuan Chen, Jie Liu, Philip Loziuk, Jina Song, et al. 2015. â4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit 4-Coumaric Acid:Coenzyme a Ligases and Modulate Metabolic Flux for 3-Hydroxylation in Monolignol Biosynthesis of Populus Trichocarpa.â Molecular Plant 8 (1): 176â87. 67 https://doi.org/10.1016/j.molp.2014.12.003. Lindermayr, Christian, Britta Möllers, Judith Fliegmann, Annette Uhlmann, Friedrich Lottspeich, Harald Meimberg, and Jürgen Ebel. 2002. âDivergent Members of a Soybean (Glycine Max L.) 4-Coumarate:Coenzyme A Ligase Gene Family.â European Journal of Biochemistry / FEBS 269 (4): 1304â15. https://doi.org/2775 [pii]. Liu, Xin Yan, Ping Ping Wang, Yi Feng Wu, Ai Xia Cheng, and Hong Xiang Lou. 2018. âCloning and Functional Characterization of Two 4-Coumarate: CoA Ligase Genes from Selaginella Moellendorffii.â Molecules 23 (3): 1â11. https://doi.org/10.3390/molecules23030595. Lyu, Xiaomei, Kuan Rei Ng, Jie Lin Lee, Rita Mark, and Wei Ning Chen. 2017. âEnhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces Cerevisiae.â Journal of Agricultural and Food Chemistry 65 (31): 6638â46. https://doi.org/10.1021/acs.jafc.7b02507. Martí, Maricarmen, Gianfranco Diretto, Verónica Aragonés, Sarah Frusciante, Oussama Ahrazem, Lourdes Gómez-Gómez, and José Antonio Daròs. 2020. âEfficient Production of Saffron Crocins and Picrocrocin in Nicotiana Benthamiana Using a Virus-Driven System.â Metabolic Engineering 61 (July): 238â50. https://doi.org/10.1016/j.ymben.2020.06.009. Martin, Cathie, Hongwei Xue, Yang Zhang, Gang Wang, Qing Zhao, Jing-Ke Weng, Xiao-Ya Chen, and Lionel Hill. 2016. âA Specialized Flavone Biosynthetic Pathway Has Evolved in the Medicinal Plant, Scutellaria Baicalensis.â Science Advances 2 (4): 1â15. https://doi.org/10.1126/sciadv.1501780. Nair, R. B., Qun Xia, Cyril J. Kartha, Eugen Kurylo, Rozina N. Hirji, Raju Datla, and Gopalan Selvaraj. 2002. âArabidopsis CYP98A3 Mediating Aromatic 3-Hydroxylation. 68 Developmental Regulation of the Gene, and Expression in Yeast.â Plant Physiology 130 (1): 210â20. https://doi.org/10.1104/pp.008649. Nakayama, Toru, Seiji Takahashi, and Toshiyuki Waki. 2019. âFormation of Flavonoid Metabolons: Functional Significance of Protein-Protein Interactions and Impact on Flavonoid Chemodiversity.â Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.00821. Newman, David J., and Gordon M. Cragg. 2020. âNatural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019.â Journal of Natural Products 83 (3): 770â803. https://doi.org/10.1021/acs.jnatprod.9b01285. Nieter, Annabel, Paul Haase-Aschoff, Sebastian Kelle, Diana Linke, Ulrich Krings, Lutz Popper, and Ralf G. Berger. 2015. âA Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago Maydis.â Applied and Environmental Microbiology 81 (5): 1679â88. https://doi.org/10.1128/AEM.02911-14. Niggeweg, Ricarda, Anthony J. Michael, and Cathie Martin. 2004. âEngineering Plants with Increased Levels of the Antioxidant Chlorogenic Acid.â Nature Biotechnology 22 (6): 746â54. https://doi.org/10.1038/nbt966. Obata, Toshihiro. 2019. âMetabolons in Plant Primary and Secondary Metabolism.â Phytochemistry Reviews 18 (6): 1483â1507. https://doi.org/10.1007/s11101-019-09619-x. Pichersky, Eran, and Robert A. Raguso. 2018. âWhy Do Plants Produce so Many Terpenoid Compounds?â New Phytologist 220 (3): 692â702. https://doi.org/10.1111/nph.14178. R Core Team (2020). 2020. âR: A Language and Environment for Statistical Computing.â R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 69 Reed, James, and Anne Osbourn. 2018. âEngineering Terpenoid Production through Transient Expression in Nicotiana Benthamiana.â Plant Cell Reports 37 (10): 1431â41. https://doi.org/10.1007/s00299-018-2296-3. Reumann, Sigrun. 2004. âSpecification of the Peroxisome Targeting Signals Type 1 and Type 2 of Plant Peroxisomes by Bioinformatics Analyses.â Plant Physiology 135 (2): 783â800. https://doi.org/10.1104/pp.103.035584. Saballos, Ana, Scott E. Sattler, Emiliano Sanchez, Timothy P. Foster, Zhanguo Xin, Chulhee Kang, Jeffrey F. Pedersen, and Wilfred Vermerris. 2012. âBrown Midrib2 (Bmr2) Encodes the Major 4-Coumarate: Coenzyme A Ligase Involved in Lignin Biosynthesis in Sorghum (Sorghum Bicolor (L.) Moench).â Plant Journal 70 (5): 818â30. https://doi.org/10.1111/j.1365-313X.2012.04933.x. Schnabel, Arianne, Fernando Cotinguiba, Benedikt Athmer, Changqing Yang, Bernhard Westermann, Angela Schaks, Andrea Porzel, Wolfgang Brandt, Frank Schumacher, and Thomas Vogt. 2019. âA Piperic Acid CoA Ligase Produces a Putative Precursor of Piperine, the Pungent Principle from Black Pepper Fruits.â Plant Journal 102 (3): 569â81. https://doi.org/10.1111/tpj.14652. Schoch, Guillaume, Simon Goepfert, Marc Morant, Alain Hehn, Denise Meyer, Pascaline Ullmann, and Danièle Werck-Reichhart. 2001. âCYP98A3 from Arabidopsis Thaliana Is a 3â-Hydroxylase of Phenolic Esters, a Missing Link in the Phenylpropanoid Pathway.â Journal of Biological Chemistry 276 (39): 36566â74. https://doi.org/10.1074/jbc.M104047200. Schröder, Joachim. 2000. âThe Family of Chalcone Synthase-Related Proteins: Functional Diversity and Evolution.â In Recent Advances in Phytochemistry. 70 https://doi.org/10.1016/S0079-9920(00)80004-0. Shockey, Jay, and John Browse. 2011. âGenome-Level and Biochemical Diversity of the Acyl-Activating Enzyme Superfamily in Plants.â Plant Journal 66 (1): 143â60. https://doi.org/10.1111/j.1365-313X.2011.04512.x. Soubeyrand, Eric, Megan Kelly, Shea A. Keene, Ann C. Bernert, Scott Latimer, Timothy S. Johnson, Christian Elowsky, Thomas A. Colquhoun, Anna K. Block, and Gilles J. Basset. 2019. âArabidopsis 4-COUMAROYL-COA LIGASE 8 Contributes to the Biosynthesis of the Benzenoid Ring of Coenzyme Q in Peroxisomes.â Biochemical Journal 476 (22): 3521â32. https://doi.org/10.1042/BCJ20190688. Srividya, Narayanan, Iris Lange, Michael Hartmann, Qunrui Li, Maryam Mirzaei, and Bernd Markus Lange. 2020. âBiochemical Characterization of Acyl Activating Enzymes for Side Chain Moieties of Taxol and Its Analogs.â Journal of Biological Chemistry 295 (15): 4963â73. https://doi.org/10.1074/jbc.RA120.012663. Sun, Haiyan, Ying Li, Shengqiu Feng, Weihua Zou, Kai Guo, Chunfen Fan, Shengli Si, and Liangcai Peng. 2013. âAnalysis of Five Rice 4-Coumarate: Coenzyme A Ligase Enzyme Activity and Stress Response for Potential Roles in Lignin and Flavonoid Biosynthesis in Rice.â Biochemical and Biophysical Research Communications 430 (3): 1151â56. https://doi.org/10.1016/j.bbrc.2012.12.019. Sunstrum, Frederick G., Liu, Hannah L., Jancsik, Sharon, Madilao, Lufiani L. 2021. â4-coumaroyl-CoA ligases in the biosynthesis of the anti-diabetic metabolite montbretin Aâ PLOS ONE. 16: 1-18. doi:10.1371/journal.pone.0257478 Takeda, Yuri, Yuki Tobimatsu, Steven D. Karlen, Taichi Koshiba, Shiro Suzuki, Masaomi Yamamura, Shinya Murakami, et al. 2018. âDownregulation of P-COUMAROYL ESTER 71 3-HYDROXYLASE in Rice Leads to Altered Cell Wall Structures and Improves Biomass Saccharification.â Plant Journal. https://doi.org/10.1111/tpj.13988. Tarling, Chris A., Kate Woods, Ran Zhang, Harry C. Brastianos, Gary D. Brayer, Raymond J. Andersen, and Stephen G. Withers. 2008. âThe Search for Novel Human Pancreatic α-Amylase Inhibitors: High-Throughput Screening of Terrestrial and Marine Natural Product Extracts.â ChemBioChem 9 (3): 433â38. https://doi.org/10.1002/cbic.200700470. Tohge, Takayuki, Federico Scossa, and Alisdair R. Fernie. 2015. âIntegrative Approaches to Enhance Understanding of Plant Metabolic Pathway Structure and Regulation.â Plant Physiology. https://doi.org/10.1104/pp.15.01006. Vanholme, Ruben, Igor Cesarino, Katarzyna Rataj, Yuguo Xiao, Lisa Sundin, Geert Goeminne, Hoon Kim, et al. 2013. âCaffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis.â Science 341 (6150): 1103â6. https://doi.org/10.1126/science.1241602. Vogt, Thomas. 2010. âPhenylpropanoid Biosynthesis.â Molecular Plant 3 (1): 2â20. https://doi.org/10.1093/mp/ssp106. Vries, Lisanne de, Marlies Brouckaert, Alexandra Chanoca, Hoon Kim, Matthew R. Regner, Vitaly I. Timokhin, Yi Sun, et al. 2021. âCRISPR-Cas9 Editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 Shows Their Importance and Partial Redundancy in Lignification in Populus Tremula x P. Alba.â Plant Biotechnology Journal. https://doi.org/https://doi.org/10.1111/pbi.13651. Williams, Leslie K., Xiaohua Zhang, Sami Caner, Christina Tysoe, Nham T. Nguyen, Jacqueline Wicki, David E. Williams, et al. 2015. âThe Amylase Inhibitor Montbretin A Reveals a New Glycosidase Inhibition Motif.â Nature Chemical Biology 11 (9): 691â96. 72 https://doi.org/10.1038/nchembio.1865. Xu, Bin, Luis L. Escamilla-Treviño, Noppadon Sathitsuksanoh, Zhengxing Shen, Hui Shen, Y. H. Percival Zhang, Richard A. Dixon, and Bingyu Zhao. 2011. âSilencing of 4-Coumarate: Coenzyme A Ligase in Switchgrass Leads to Reduced Lignin Content and Improved Fermentable Sugar Yields for Biofuel Production.â New Phytologist 192 (3): 611â25. https://doi.org/10.1111/j.1469-8137.2011.03830.x. Yonekura-Sakakibara, Keiko, and Kazuki Saito. 2009. âFunctional Genomics for Plant Natural Product Biosynthesis.â Natural Product Reports. https://doi.org/10.1039/b817077k. Yuen, Violet G., John Coleman, Steven G. Withers, Raymond J. Andersen, Gary D. Brayer, Sally Mustafa, and John H. McNeill. 2016. âGlucose Lowering Effect of Montbretin A in Zucker Diabetic Fatty Rats.â Molecular and Cellular Biochemistry 411 (1â2): 373â81. https://doi.org/10.1007/s11010-015-2599-4. 73 Appendices Appendix A A.1 Protein accession numbers. AAEs that were used in the phylogenetic analysis are listed. AAEs that were also used in the BLASTP search of the montbretia corm time-course transcriptome are bold. Name Clade Organism Accession AtLACS1 I Arabidopsis thaliana O22898.1 At4CL1 IV Arabidopsis thaliana NP_175579.1 At4CL3 IV Arabidopsis thaliana NP_176686.1 Gm4CL1 IV Glycine max AAL98709.1 Gm4CL2 IV Glycine max NP_001236418.1 Gm4CL3 IV Glycine max NP_001237270.1 Os4CL1 IV Oryza sativa XP_015650724.1 Os4CL2 IV Oryza sativa XP_015624111.1 Pv4CL1 IV Panicum virgatum ACD02135.1 Pv4CL2 IV Panicum virgatum ADZ96250.1 AtOPCL1 V Arabidopsis thaliana Q84P21.2 AtACS5 V Arabidopsis thaliana Q9LQ12.1 Pn4CL2 V Piper nigrum MH078049 SbCLL7 V Scutellaria baicalensis AMW91733.1 AtACOS5 V Arabidopsis thaliana OAP13468.1 At4g19010 V Arabidopsis thaliana OAP13468.1 AtBZO1 VI Arabidopsis thaliana Q9SS01.1 AtAAE7 VI Arabidopsis thaliana Q8VZF1.1 HcCNL VI Hypericum calycinum AFS60176.1 AtAAE13 VII Arabidopsis thaliana NP_001319565.1 AtAAE14 VII Arabidopsis thaliana AEE31239.1 AtAAE3 VII Arabidopsis thaliana Q9SMT7.1 A.2 Contig names and PTS1 tripeptide if present. Contig name Gene name PTS1 tripeptide sequence yc_T2_E1.L.20183 CcAAE1 PKL 74 yc_T1_E0.L.218594 CcAAE2 (Cc4CL1) yc_T1_E1.S.88724 CcAAE3 ARL yc_T2_E1.L.56472 CcAAE4 yc_T1_E1.L.30829 CcAAE5 yc_T6_E1.S.85585 CcAAE7 PKL yc_T1_E0.L.209455 CcAAE9 yc_T1_E1.L.29230 CcAAE10 (Cc4CL2) yc_T2_E1.L.51517 CcAAE6 CRI yc_T1_E1.L.48553 CcAAE8 SKM yc_T1_E2.L.11983 CcAAE11 SKM yc_T2_E1.L.52762 CcAAE12 SKM yc_T3_E0.L.155683 CcAAE13 SRL yc_T3_E1.L.29491 CcAAE14 SKL yc_T3_E2.L.12319 CcAAE15 SKL yc_T4_E1.L.25973 CcAAE16 75 A.3 Correlation of CcAAEs expression with previously identified MbA pathway genes during young corm development. Pearson correlation coefficients are given and selected candidate genes are bold. Name max correlation CcUGT1 CcUGT2 CcUGT3 CcUGT4 CcUGT5 CcAT CcF3H-2 CcF3H-1 CcFLS1 CcCYP2 CcCYP1 CcAAE2 (Cc4CL1) 0.98 0.98 0.95 0.95 0.95 0.98 0.88 0.90 0.28 0.95 0.93 0.96 CcAAE10 (Cc4CL2) 0.72 0.56 0.62 0.70 0.52 0.55 0.36 0.32 0.22 0.72 0.36 0.56 CcAAE1 0.93 0.93 0.90 0.85 0.92 0.90 0.76 0.87 0.31 0.85 0.91 0.87 CcAAE3 0.91 0.78 0.69 0.62 0.76 0.79 0.83 0.91 0.27 0.62 0.91 0.79 CcAAE4 0.99 0.86 0.79 0.81 0.81 0.90 0.99 0.91 0.24 0.82 0.88 0.90 CcAAE5 0.94 0.89 0.94 0.94 0.89 0.87 0.66 0.66 0.17 0.94 0.71 0.77 CcAAE7 0.97 0.96 0.92 0.90 0.95 0.97 0.95 0.93 0.15 0.89 0.92 0.90 CcAAE9 0.99 0.91 0.84 0.86 0.84 0.94 0.99 0.92 0.25 0.88 0.91 0.98 CcAAE6 0.55 -0.02 -0.03 0.09 -0.11 0.01 0.14 0.05 0.55 0.09 0.02 0.08 CcAAE8 0.29 -0.64 -0.75 -0.69 -0.74 -0.59 -0.28 -0.31 0.29 -0.66 -0.37 -0.37 CcAAE11 0.00 -0.34 -0.43 -0.54 -0.31 -0.35 -0.21 -0.06 0.00 -0.56 -0.07 -0.33 CcAAE12 0.10 0.09 0.09 -0.04 0.19 0.08 0.06 0.10 -0.55 -0.07 0.10 -0.05 CcAAE13 -0.47 -0.93 -0.89 -0.87 -0.89 -0.92 -0.81 -0.91 -0.47 -0.87 -0.94 -0.91 CcAAE14 0.26 -0.83 -0.85 -0.86 -0.82 -0.84 -0.72 -0.6 0.26 -0.88 -0.61 -0.79 CcAAE15 -0.33 -0.98 -0.97 -0.93 -0.97 -0.96 -0.83 -0.91 -0.33 -0.93 -0.93 -0.89 CcAAE16 -0.29 -0.60 -0.67 -0.68 -0.60 -0.56 -0.29 -0.38 -0.29 -0.68 -0.44 -0.51 76 A.4 Expression data of 16 candidate AAEs. Average counts per million (cpm) of two biological replicates (n=2) see Irmisch et al., 2020. Contig number AAE 10-Jun 27-Jun 22-Jul 16-Aug 12-Sep 06-Oct yc_T2_E1.L.20183 CcAAE1 11.88 9.71 10.20 4.10 7.17 5.06 yc_T1_E1.L.29230 CcAAE10 (Cc4CL2) 61.36 39.16 26.44 24.78 46.68 38.97 yc_T1_E2.L.11983 CcAAE11 9.90 16.61 23.41 18.09 14.72 15.52 yc_T2_E1.L.52762 CcAAE12 2.06 2.08 3.39 3.05 1.69 1.16 yc_T3_E0.L.155683 CcAAE13 24.84 40.48 53.06 128.48 80.65 97.37 yc_T3_E1.L.29491 CcAAE14 0.27 2.52 4.69 4.41 3.75 7.88 yc_T3_E2.L.12319 CcAAE15 18.25 23.90 26.98 40.22 35.74 36.46 yc_T4_E1.L.25973 CcAAE16 9.00 23.62 22.04 29.92 18.98 20.50 yc_T1_E0.L.218594 CcAAE2 (Cc4CL1) 89.68 78.05 58.93 23.13 47.15 26.68 yc_T1_E1.S.88724 CcAAE3 6.40 8.74 8.27 3.81 4.01 2.77 yc_T2_E1.L.56472 CcAAE4 1.21 1.87 0.71 0.46 0.32 0.21 yc_T1_E1.L.30829 CcAAE5 5.46 2.92 2.14 0.93 2.05 1.67 yc_T2_E1.L.51517 CcAAE6 4.30 5.17 2.80 3.72 4.00 5.81 yc_T6_E1.S.85585 CcAAE7 3.61 3.75 2.92 2.13 1.88 1.68 yc_T1_E1.L.48553 CcAAE8 8.27 14.83 12.35 13.52 14.41 14.85 yc_T1_E0.L.209455 CcAAE9 8.46 9.77 6.84 5.65 6.53 5.04 77 A.5 Activity and expression of AAEs. (A) Activity of heterologously expressed AAEs with different hydroxycinnamic acid substrates. (B) Protein gel blot showing AAE expression. + enzyme activity detected, - no enzyme activity detected A Cinnamic acid p-coumaric acid Caffeic acid Ferulic acid Sinapic acid Cc4CL1 - + + + - Cc4CL2 + + + + - CcAAE1 - - - - - CcAAE3 - - - - - CcAAE4 - - - - - CcAAE5 - - - - - CcAAE7 - - - - - CcAAE9 - - - - - B CcAAE2 (Cc4CL1) CcAAE10 (Cc4CL2) CcAAE3 CcAAE4 CcAAE5 CcAAE9 CcAAE7 kDa 75 50 37 75 50 37 kDa CcAAE1 78 A.6 Combinations of genes used for transient expression in N. benthamiana. Each gene was individually expressed as a 35Spro:gene construct and genes were combined by mixing equal volumes of Agrobacterium cultures for infiltration. Combination Constructs MBG CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 MBG+Cc4CL1 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + Cc4CL1 MBG+Cc4CL2 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + Cc4CL2 MBG+CcAAE1 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE1 MBG+CcAAE3 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE3 MBG+CcAAE4 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE4 MBG+CcAAE5 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE5 MBG+CcAAE7 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE7 MBG+CcAAE9 CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcUGT3 + CcUGT4 + CcUGT5 + CcAAE9 79 A.7 Montbretin A, B and C levels in N. benthamiana plants transiently expressing MBGs and AAE. Mean (n = 4) and SE are shown. See appendix A.6 for combination of genes used for infiltration. An ANOVA was used to test for significant differences. MbA (F8,18 = 1.57, p = 0.20), MbB (F8,18 = 1.43, p = 0.25), MbC (F8,18 = 1.06, p = 0.43) 0 100 200 300 400 MBGs MBGs + Cc4CL1 MBGs + Cc4CL2 MBGs + CcAAE1 MBGs + CcAAE3 MBGs + CcAAE4 MBGs + CcAAE5 MBGs + CcAA7 MBGs + CcAAE9 µg/g (FW) MbA MbB MbC 80 A.8 Identification of montbretins produced by N. benthamiana transiently expressing MBGs. (A) Extracted ion chromatogram (EIC) of MbA (peak 2, m/z 1227.5), MbB (peak 1, m/z 1211.5), and MbC (peak 3, 1241.5) MS/MS fragmentation patterns of MbA (B), MbB (C), MbC (D) and MS3 of MbC (m/z 1241.5 Ã 963.0) (E). MS/MS 1211.5 A 10 Relative intensity (EIC x 100 000) 1 3 1 3 5.0 6.0 7.0 8.0 9.0 Retention Time (min) EIC 1227.5 EIC 1211.5 EIC 1241.5 20 1 2 3 787 0 2 4 6 Relative intensity (EIC x 100,000) 200 600 1000 m/z MS/MS 963.0 B Relative intensity (EIC x 100,000) 787 949 1065.4 0.0 0.2 0.5 0.7 1.0 1.2 400 800 1200 m/z MS/MS 1227.5 MbA â MbB â Relative intensity (EIC x 100,000) 787 933 1065 0 2 4 6 200 600 1000 m/z â C Relative intensity (EIC x 10,000) 787 963 0 2 4 6 8 200 600 1000 m/z MS/MS 1241.5 MbC â D E MbC-XR 81 A.9 Parts of the MoClo system (Addgene, MA, USA) used for the construction of S. cerevisiae expression modules. Modules were assembled using the Golden-gate cloning pipeline described by Lee and co-workers (2015). Position 1 spacer, ConLS, ConR1 Position 2 CcCHS2, ConL1, ConR2, pHTB2, tENO1 Position 3 CcCHI2, ConL2, ConR3, pRPL18B, tSSA1 Position 4 4CL, ConL3, ConR4, pHHF1, tTDH1 Position 5 spacer, ConL4, ConRE Vector backbone ConLS', ConRE', CEN6/ARS4, Kan-ColE1, URA3 A.10 Identification of products formed by S. cerevisiae expressing a flavanone biosynthetic module. Peak 1 and peak 3 produced by S. cerevisiae as shown in Figure 2.6 were tentatively identified based on their MS/MS fragmentation patterns compared to fragmentation patterns described by Waki et al., 2020 [1]. Peak 1, tentatively identified as p-coumaroyltriacetic acid lactone; peak; peak 3, tentatively identified as caffeoyltriacetic acid lactone. 1. Waki T, Mameda R, Nakano T, Yamada S, Terashita M, Ito K, Tenma N, Li Y, Fujino N, Uno K et al: A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity. Nature com 2020, 11(1):870. 125 145 185 201 227 1 3 75 125 175 225 m/z â MS/MS 271 Peak 1 5 â Peak 3 125 152 161 203 217 243 1 3 75 125 175 225 275 m/z MS/MS 287 Relative intensity (EIC x 1 000) 82 A.11 Oligonucleotides used in this study. Gene Forward (5â - 3â) Reverse (5â - 3â) CcAAE1 ATGTCACCCTCCATTGATCCCAAC CTACAATTTGGGATTCAATTTCGAGGA CcAAE2 (Cc4CL1) ATGATCACTATAGCAACTCATGAAGCTC TTAAGTAGAAACAACCACTCTTGCCC CcAAE3 ATGGCAGTCTTGGAAGACAATGGCT TCATAGCCTAGCTGAGATCTCTAGC CcAAE4 ATGGCAAACTTGGCCTGCTTTGATA CTACAGTTGGTCACTCCCTTTTTTATTG CcAAE5 ATGTCTTCTTCTTCCTCCATAGATCC TCAGTAGTAGAATGGTACTTCTGTGGA CcAAE7 ATGTCGCCCTCCTCCATTGAC CTACAATTTAGGATTCGTTGAGGATGCT CcAAE9 ATGGGAGAGTTCGCAGTACGTAAAG ATAGGCCTGCCTCTTGGCCAAAT CcAAE10 (Cc4CL2) ATGGGTTCCATTCCTTCGGAGAAAG TCACAGCTGCTGCCCCTTAG 83 Appendix B B.1 Montbretia phenylpropanoid biosynthesis genes >CcC3H1 ATGACCAAAGTGACAACAATGGAAGACCAATTCCTCCTCCTCCTCTCCATCTCCGTTGCT ATCGTCACCGTCCTCCTCTCCTTCCTCCTCTACCGCCGCCTGTCGGTGCGCTTACCCCCC GGTCCCCGGCCGCTGCCGGTGGTCGGCAACCTCTACGACATCAAGCCGGTCCGGTTCAGG TGCTTCGCCGATTGGGCCGGCACCTACGGACCGATATTCTCCGTCTGGTTCGGCACCACG CTCAACGTGGTCGTCTCCAGCTCTGAGCTGGCACGGGAGGTTTTGAAAGAGAACGACCAG GCCCTCGCCGACCGGCCCAGGAACCGGGCCGCCGCCCGGTTCAGCCGCGACGGCAAGGAC CTGATATGGGCCGACTATGGGCCCCACTACGTGAAGGTTAGGAAGGTGTGCAATCTCGAG CTGTTCTCGCCGAAGCGGCTCGAGGCGCTCCGACCCATCAGGGAGGACGAGGTCACCGCC ATGGTCGACTCCATCTTCAAAGAATGCACTCAACCAGAAAAGAACGGGAAGAGCTTGGTA GTGAGGGAGCACATTTCCGGGGTGGCGTTCAACAACATTACACGACTTGCATTCGGAAAG CGCTTTGTGAATTCAGAAGGCGTGATGGACGAACAAGGCGTGGAATTCAAGGCAATTGTG GCCAATGGACTCAAATTTGGCGCGTCGCTCTCCTTGGGAGAGTATGTGAATTGGCTACGC TGGATGTATCCTCTAGACGAAGAGGCCTTTGCCAAGCACGGTGCTAGGAGAGATCGTCTC ACCAGAATCATAATGGAGGAGCATACGCGCGCTCGCAAGGAGAGTGGTGGTGTGAGGCAA CACTTTGTCGATGCGCTGCTCACCCTTCAGGAGAAGTATGACCTCAGTGAGGATACCATT ATCGGCCTTCTTTGGGATATGATTACTGCAGGAATGGACACAACTGTCATCACTGTGGAA TGGGCCATGGCCGAGCTAGTTAGAAACCCCCGAGTCCAACAGAAGGTCCAAGAAGAACTA GACAGAGTCATAGGCACAGACCGCATCCTGACCGAAGCCGACTTCTCGAGCCTCCCTTAT CTCCACTGCATCGTCAAGGAGTCCCTCCGTCTCCACCCGCCAACGCCACTCATGCTCCCC CACAAGGCGACTGCCAATGTGAAGATCGGAGGCTACGACATCCCCAAAGGATCCAACGTT CATGTCAATGTGTGGGCAATAGCTCGCGATCCCAAAGTGTGGAAGAATCCGCTGGAGTTC CGCCCCGAGCGGTTTGAAGAGGAGAATATTGACATCAAGGGGAACGACTTCCGTGTGCTG CCGTTCGGTGCCGGTCGAAGGGTGTGCCCTGGGGCACAATTAGGCATCAACTTGGTGCAG TCGATGCTCGGCCACCTCTTGCATCATTTTGAGTGGAAGCTGCCCAAAGGAGTGAAACTG GAGGAAGTTGACATGTCGGAGAATCCCGGAATGGTTACTTTCATGCACACGCCTCTGCAA GCTATTGCGGTACCTCGACTGCCGTCACAACTGTACAACCGTGTCGCTGCCGATATGTAA >CcHCT3 ATGGCGACGGTGGAGATAACAGTGAGGGACACGTCGATGGTGGTCCCGGCGGAGGAGACG CCTCGACGGAGGCTGTGGAACTCGAACCTGGACCTGGTGGTCCCCAGGTTCCACACCCCC AGCGTCTACTTCTACCGCCGGCCCGCGGACGCCGCGGGGGAATCGTTCTTCGATGCGGCG GTGCTGAAGGATGCGCTGAGCCGGGCACTGGTGCCGTTCTATCCCATGGCCGGCAGGCTG GCCAGGGACGAGGACGGACGCATCGAGATCGACTGCCAGGGACAGGGGGTGCTCTTGGTC GTCGCCGAGACGGAATCTCTCGTTGACGATTTCGGCGATTTTGCTCCCACTGTGGAGATG AAGAAGCTCATTCCTACTGTGGACTATACCGACGACATTTCGTCTTATCCTCTCTTAGTG CTGCAGGTAACCCACTTCAAGTGCGGAGGTGCATCTTTGGGTGTGGGCATGCAGCACCAT GTTGCCGACGGCACCTCTGGCCTCCACTTCATCAACTCATGGTCCGATGTTGCACGTGGC GTTGGCATCTCCGTCCTTCCCTTCATCGACCGCAATCTCCTCCGCGCGCGTGACCCTCCC TCCCCCTCCTTCCCTCACATTGAGTACCAGCCTGCCCCCCCAATGAAGCAGCAGCTAGCC ATACCAACACCAACTACTGTCAAGTCTGGCGATGTTGCGGCTTCTGTTCGCTTGTTCAAA CTCAGGCCTGAACAAGTTAACCTCCTGAAATTGAAGCTCAAAGCGAGGTACAGCTCGTAT GCCTTTATCGCTGGCCACGTCTGGCGTTGTGCCTGCATTGCCCGTGAGCTTCCCGAGGAC CAGCCAACCAAACTATACTGTGCAACCGATGGTCGAACTCGCCTTGAGCCGCCATTACCA GAAGGGTACTTTGGGAACGTGATCTATACTGCGACGCCAATCGCAGAAGCAGGTGAGATG GCGGGCAATGACGGCTTAGAGGTGGCGGCAGAGAGGATACAATCAGCATTGACAAGGATG GCCGGCGAGTACATGCGATCAGCACTGGACTATTTGGAGCTGCAGCCTGATCTTAGCAAG TTGGTGAGAGGAGCACACACGTTCAAGTGTCCGAACATTGGGATCACTAGCTGGACCAGG 84 TTGCCAATTCACGATGCGGACTTTGGTTGGGGAAGGCCCATTTTCATGGGACCTCCATGG ATTTCATACGAAGGGTTGGCTTTCATATTGCCTAGTAGCCAAGGGGATAGGAGCGTGTCG GTGGTAATCGCTTTGCAGCCTGAGCATATGGTTAGATTTGAAAAGTTGCTCTATGACTTG TAG >CcCSE2 ATGCCGACAAAATACTTCTGGGGCGACGAGCCCGAAGAATCCGAGTACTATGCCTCCCAT GGCGTCTCCCACTCTCAGTCCTACTTCACCACTCCCGCCGGCCCCAAGCTCTTCACCCAG TCCTTCGCCCCTCTCGGCCGACAACCCAAAGCTCTCGTCTTTATGACCCACGGCTACGGC TCCGACTCCGGCTGGCTCTTCCAGAAGATCGCCATCGCCTTCGCCTCCTGGGGCTACTCC GTCCACTGCGCCGACCTCCTCGGCCACGGCCGCTCCGACGGCATCCACGGCTACCTCGGC GACCTCCAGGCCGTCGCCGCCGCCTCCCTCTCCTTCTTCCTCTCCGTCATCAAGAATCAT CATGGAGACGATCTCCCCGCCTTCCTCTTCGGCGAGTCGCTCGGCGGCCTCGTCACCCTG CTCGCCTACTTCCAGTCCCCGCCCGACGTCTGGAGCGGCGTCATCCTCTCCGCGCCGCTC TTCGTCATCCCGGAGGACATGAAGCCGTCAAAGCTCCGCCTTTTCCTCTTCGGCCTGCTC TTCGGCGCGGCAGACACGTGGCAGATGATGCCGGAGAACAACATGGTCGGGAAGGCGATA CGCGACCCGGAGCGGCTGAAGATTATCGCCTCCAACCCGAGGCGGTACACCGGTCGGCCC CGGGTCGGGACGATGCGGGAGATCGCGCGGATGTGCGAGCACGTGCAGGCCAATTTCGAC AAGGTGACGGCGCCGTTCCTGGTGCTGCACGGGACGGACGACGGGGTGACGGCGCCGGAG GGGTCGAAGATGCTTTACGAGAAGGCGGCGAGCGAGGACAAGGAGCTTGTTTTGTATGAG GGGATGCTGCATTCGCTTGTGCAGGGGGAGCCGGAGGAGAACTGCCAGCGGGTGCTGGCG GATATGAGGGCGTGGATTGACAAGAGGGTGGTGGACAAGTGA B.2 Optimal parameters for xcms as determined by the âIPOâ package. LC-QTOF data for the four samples expressing MBGs were used for parameter optimization. Parameter (peak picking) Optimal value Parameter (Retention time correction) Optimal value Snthresh 4 gapInit 0.22 noise 0 gapExtent 2.7 Value_of_prefilter 100 profStep 0 Min_peakwidth 20.4 bw 5 Max_peakwidth 70 minfrac 0.7 ppm 21 mzwid 0.01 mzdiff -9.8x10-5 distFunc âcor_optâ prefilter 3 response 1 mzCenterFun âwMeanâ factorDiag 2 integrate 1 factorGap 1 localAlignment 0 retcorMethod âorbiwarpâ minsamp 1 max 50 center 1 85 B.3 Additional combinations of genes used for transient expression in N. benthamiana. Each gene was individually expressed as a 35Spro:gene construct and genes were combined by mixing equal volumes of Agrobacterium cultures for infiltration. Statistical analysis was not performed since yield increases were not observed. Experiment Combinations [Mini-MbA] (µg/g FW) A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT 10.0 ± 3.4 A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcCSE2 119.8 ± 95.0 A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcCSE2 + CcC3H1 61.5 ± 29.6 A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcHCT3 60.6 ± 45.3 A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcHCT3 + CcCSE2 57.1 ± 26.7 A CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcHCT3 + CcC3H1 55.5 ± 13.7 B CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT 81.5 ± 21.5 B CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + UmChlE 148.4 ± 33.0 B CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + UmChlE + Cc4CL1 95.1 ± 11.2 B CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + UmChlE + Cc4CL2 137.5 ± 36.4 C CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT 102.0 ± 13.2 C CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcC4H1 87.8 ± 14.9 C CcFLS + CcCYP2 + CcMYB4 + CcUGT1 + CcUGT2 + CcAT + CcC4H1 + CcC3H1 56.2 ± 4.2
Thesis/Dissertation
eng
Genome Science and Technology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Bohlmann, Jörg
Engineering montbretin A biosynthesis in Nicotiana benthamiana
Text