Science, Faculty of
DSpace
UBCV
Haghighat Kashani, Amir
Master of Science - MSc
University of British Columbia
Capsaicinoids are a class of compounds natively produced by pepper plants as secondary metabolites. They possess therapeutic properties and have been used for treating medical conditions such as chronic pain, overactive bladder and diabetes. Capsaicin, a well-known chemical in this class, is responsible for the spicy taste of chili peppers. The precursors for capsaicinoids include vanillylamine and an acyl-CoA. Capsinoids, a group of capsaicinoid analogues, use vanillyl alcohol as a precursor instead of vanillylamine, making them less pungent. Here, we describe preliminary work towards the development of an E. coli strain for the production of capsaicinoids and capsinoids using vanillin and glycerol as precursors. Successful implementation of this system could allow targeted production of compound libraries or individual compounds if required. Such flexibility can be achieved through the mixing and matching of different enzymes within the host system. This platform can lead to production of high-value low-volume chemical products that have pharmaceutical applications. However, a sufficiently productive platform could also provide a less resource intensive source of capsaicinoids, currently gathered from pepper plants at less than 1% yield. In this work, we evaluate E. coli as a host for the synthesis of capsaicinoids and capsinoids. Initially, we created a pathway and successfully demonstrated synthesis of C6 to C12 straight and branched-chain fatty acids. We then focused on candidate enzymes for acyl-CoA synthesis to activate the aforementioned fatty acids, and a number of potential candidates were identified based on literature review. Furthermore, we investigated vanillylamine and vanillyl alcohol production. Previous publications have demonstrated conversion of vanillin to vanillylamine in E. coli using transaminases. Vanillyl alcohol on the other hand can be synthesized from vanillin using the endogenous reductases in E. coli. Lastly, we attempted expression of capsaicin synthase in E. coli with and without the SUMO tag, however results were inconclusive. More investigation needs to be performed to determine whether capsaicin synthase, a key enzyme on our pathway, can be expressed in its active form.
EVALUATING E. COLI AS A CHASSIS FOR THE PRODUCTION OF CAPSAICINOIDS AND CAPSINOIDS by Amir Haghighat Kashani BComm., The University of British Columbia, 2016 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 © Amir Haghighat Kashani, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Evaluating E. coli as a Chassis for the Production of Capsaicinoids and Capsinoids submitted by Amir Haghighat Kashani in partial fulfillment of the requirements for the degree of Master of Science in Genome Science and Technology Examining Committee: Dr. Vikramaditya G. Yadav, Chemical and Biological Engineering, UBC Supervisor Dr. Harry Brumer, Chemistry, UBC Supervisory Committee Member Dr. Katherine S. Ryan, Chemistry, UBC Supervisory Committee Member iii Abstract Capsaicinoids are a class of compounds natively produced by pepper plants as secondary metabolites. They possess therapeutic properties and have been used for treating medical conditions such as chronic pain, overactive bladder and diabetes. Capsaicin, a well-known chemical in this class, is responsible for the spicy taste of chili peppers. The precursors for capsaicinoids include vanillylamine and an acyl-CoA. Capsinoids, a group of capsaicinoid analogues, use vanillyl alcohol as a precursor instead of vanillylamine, making them less pungent. Here, we describe preliminary work towards the development of an E. coli strain for the production of capsaicinoids and capsinoids using vanillin and glycerol as precursors. Successful implementation of this system could allow targeted production of compound libraries or individual compounds if required. Such flexibility can be achieved through the mixing and matching of different enzymes within the host system. This platform can lead to production of high-value low-volume chemical products that have pharmaceutical applications. However, a sufficiently productive platform could also provide a less resource intensive source of capsaicinoids, currently gathered from pepper plants at less than 1% yield. In this work, we evaluate E. coli as a host for the synthesis of capsaicinoids and capsinoids. Initially, we created a pathway and successfully demonstrated synthesis of C6 to C12 straight and branched-chain fatty acids. We then focused on candidate enzymes for acyl-CoA synthesis to activate the aforementioned fatty acids, and a number of potential candidates were identified based on literature review. Furthermore, we investigated vanillylamine and vanillyl alcohol production. Previous publications have demonstrated conversion of vanillin to vanillylamine in E. coli using transaminases. Vanillyl alcohol on the other hand can be synthesized from vanillin using the endogenous reductases in E. coli. Lastly, we attempted expression of capsaicin synthase in E. coli with and without the SUMO tag, however results were inconclusive. More investigation needs to be performed to determine whether capsaicin synthase, a key enzyme on our pathway, can be expressed in its active form. iv Lay Summary Chili peppers have been incorporated into many cuisines and hot sauces. The core of their spicy taste is produced by a compound named capsaicin, and this molecule belongs to larger family of compounds called capsaicinoids. These molecules have applications in cosmetic products and pepper sprays, as well in medical field such as treating chronic pain, diabetes, and weight loss. Considerable amount of resources have to be used to grow pepper plants in large quantities just to obtain small amounts of capsaicinoids. A more efficient production method can be developed by inserting pepper plant DNA into microorganisms like bacteria to make them produce capsaicinoids. Reducing greenhouse emissions and production times are some of the major advantages of this approach. This project aims to create a proof of concept for making capsaicinoids in engineered microorganisms by feeding them sugar and vanillin, the principal compound that gives vanilla its flavor and aroma. v Preface This project was jointly designed by Amir Kashani and Dr. Vikramaditya G. Yadav. All experiments, data collection and analysis were performed by Amir Kashani under the supervision of Dr. Vikramaditya G. Yadav. vi Table of Contents Abstract ............................................................................................................................................ iii Lay Summary .................................................................................................................................... iv Preface .............................................................................................................................................. v Table of Contents .............................................................................................................................. vi List of Figures .................................................................................................................................... ix List of Abbreviations .......................................................................................................................... x Acknowledgments ............................................................................................................................ xi Chapter 1 Introduction .......................................................................................................................1 1.1 Introduction to Capsaicinoids and Capsinoids ........................................................................... 1 1.2 Medical Studies and Industrial Applications .............................................................................. 3 1.3 TRPV1 Receptor and its Relation to Pungency .......................................................................... 6 1.4 Evolution of Capsicum Species and Capsaicinoid Biosynthesis Pathway .................................. 7 1.5 Objectives of this Study ........................................................................................................... 10 1.6 Potential Benefits for Microbial Production of Capsinoids and Capsaicinoids ........................ 15 Chapter 2 Biosynthesis of Medium Straight and Branch-Chain Fatty Acids ......................................... 16 2.1 Introduction ............................................................................................................................. 16 2.1.1 Straight-chain Fatty Acid Synthesis and Regulation ....................................................... 16 2.1.2 Thioesterase Acyl Chain Length Selectivity and Engineering ......................................... 17 2.1.3 Fatty Acid Chain Elongation Regulation beyond the TE ................................................. 19 2.1.4 Fatty Acid Production Titers ........................................................................................... 20 2.1.5 Branched-chain Fatty Acid Synthesis ............................................................................. 22 2.1.6 Thioesterase Candidates ................................................................................................ 23 2.2 Materials and Methods ............................................................................................................ 24 2.2.1 Expression Vectors ......................................................................................................... 24 2.2.2 Designing and Cloning Constructs .................................................................................. 25 2.2.3 Fatty Acid Production and Analysis ................................................................................ 27 2.3 Results ...................................................................................................................................... 28 2.4 Discussion................................................................................................................................. 31 2.5 Future Work ............................................................................................................................. 33 2.6 Conclusion ................................................................................................................................ 34 Chapter 3 Identifying a Suitable Acyl-CoA Synthase .......................................................................... 35 3.1 Introduction ............................................................................................................................. 35 3.1.1 Acyl-CoA Synthase Chain Length Selectivity and Engineering ....................................... 36 vii 3.1.2 Evaluated ACSs in E. coli and Challenges in ACS Comparisons ...................................... 37 3.1.3 E. coli-based screen for Acyl-CoA Synthase Specificity .................................................. 39 3.1.4 Acyl-CoA Synthase Candidates ....................................................................................... 40 3.2 Materials and Methods ............................................................................................................ 41 3.2.1 Designing and Cloning Constructs .................................................................................. 41 3.2.2 Protein Expression and SDS-PAGE ................................................................................. 42 3.3 Results and Discussion ............................................................................................................. 43 3.4 Future work .............................................................................................................................. 44 3.5 Conclusion ................................................................................................................................ 45 Chapter 4 Production of Vanillylamine and Vanillyl Alcohol............................................................... 46 4.1 Introduction ............................................................................................................................. 46 4.1.1 Production of Vanillylamine and Enzyme Candidates ................................................... 46 4.1.2 Production of Vanillyl alcohol ........................................................................................ 48 4.2 Materials and Methods ............................................................................................................ 48 4.2.1 Designing and Cloning Constructs .................................................................................. 48 4.2.2 Protein Expression and SDS-PAGE ................................................................................. 49 4.3 Results and Discussion ............................................................................................................. 49 4.4 Future Work ............................................................................................................................. 50 4.5 Conclusion ................................................................................................................................ 51 Chapter 5 Capsaicin Synthase and the Condensation Reaction .......................................................... 52 5.1 Introduction ............................................................................................................................. 52 5.1.1 BAHD Enzyme Family ..................................................................................................... 52 5.1.2 Capsaicin Synthase Solubility ......................................................................................... 55 5.2 Materials and Methods ............................................................................................................ 55 5.2.1 Designing and Cloning Constructs .................................................................................. 55 5.2.2 Protein Expression and SDS-PAGE ................................................................................. 57 5.3 Results and Discussion ............................................................................................................. 57 5.4 Future Work ............................................................................................................................. 58 5.5 Conclusion ................................................................................................................................ 59 Chapter 6 Summary and Conclusion ................................................................................................. 60 Chapter 7 Future Work ..................................................................................................................... 62 7.1 Eliminating Dependency on non-natural vanillin ..................................................................... 62 7.2 Capsaicinoid Antimicrobial Properties and Their Inhibitory Effects ........................................ 63 Bibliography .................................................................................................................................... 64 Appendices ...................................................................................................................................... 72 viii Supplementary Table 1. Primers Used in this Study ...................................................................... 72 Supplementary Table 2. Sequencing and Colony PCR Primers ...................................................... 74 Supplementary Table 3. Primers Used for BF211 Twin Primer Assembly ..................................... 75 Supplementary Table 4. Plasmids and Bacterial Strains Used in this Study .................................. 76 Supplementary Table 5. GenBank Accession Numbers and Sequences ........................................ 77 Supplementary Table 6. Fatty Acid Yield Quantification ............................................................... 82 Supplementary Figure 1. GC-MS Chromatograms ......................................................................... 83 ix List of Figures Figure 1.1 Chemical Structure of Some of the Identified Capsaicinoids and Capsinoids ............................. 2 Figure 1.2 Capsaicin Synthesis Pathway ....................................................................................................... 9 Figure 1.3 Proposed Capsaicinoid and Capsinoid Synthesis Pathway in E. coli .......................................... 14 Figure 2.1 Straight-chain FA Synthesis Pathway ......................................................................................... 17 Figure 2.2 Regulation of Fatty Acid Synthesis and Degradation ................................................................. 21 Figure 2.3 Visual Depiction of pET28D Vector ............................................................................................ 25 Figure 2.4 Visual Depiction of Fatty Acid Constructs .................................................................................. 27 Figure 2.5 FA Profile of AcTesA Constructs and Comparison with Prior Publications ................................ 30 Figure 3.1 Four Conserved Protein Regions of Acyl-CoA Synthases ........................................................... 36 Figure 3.2 Activity Rate of RevS on Saturated and Monounsaturated Medium-chain Fatty Acids ............ 38 Figure 3.3 Visual Depiction of the Four Acyl-CoA Synthase Constructs ..................................................... 42 Figure 3.4 SDS-PAGE Results from Acyl-CoA Synthase Constructs ............................................................. 43 Figure 4.1 Visual depiction of the Transaminase Constructs ...................................................................... 49 Figure 4.2 SDS-PAGE results of pAMT and CV2025 Transaminase ............................................................. 50 Figure 5.1 The Two Main Motifs of the BAHD Family ................................................................................. 54 Figure 5.2 Visual Depiction of Capsaicin Synthase Constructs ................................................................... 56 Figure 5.3 AT3 and SUMO-AT3 SDS-PAGE Results ...................................................................................... 57 x List of Abbreviations BCCS – Branched-chain α-ketoacyl-CoA Starters BCFA(s) – Branched-chain Fatty Acid(s) CC(s) – Capsaicinoid(s) and Capsinoid(s) DPA – Days Post Anthesis FA(s) – Fatty Acid(s) FFA(s) – Free Fatty Acid(s) GA – Gibson Assembly MCS – Multiple Cloning Site RBS – Ribosome Binding Site SCFA(s) – Straight-chain Fatty Acid(s) TE(s) – Thioesterase(s) TIR – Transcription Initiation Rate WT – Wild Type xi Acknowledgments I would like to thank my supervisor for providing me with resources and advice to conduct my research, my committee members for their support throughout my research, the GSAT program for their openness to allow a student with a non-traditional background pursue a master’s degree in science, and the ECOSCOPE program for funding my master’s research, and making this work possible. I also thank my labmates, Carmen Bayly, Kene Ene, Roza Ghaemi, Kevin Salim, Gaurav Subedi, and Steven Zhao for their friendship and their contributions towards creating a better workplace. Finally, a special thanks to my parents and my sister for their unconditional care and their comforting kindness. 1 Chapter 1 Introduction 1.1 Introduction to Capsaicinoids and Capsinoids Over thousands of years, humans have adapted to enjoy the burning sensation of chili peppers, enjoying them in a variety of cuisines and food related products. Early commercial hot sauces emerged in early 19th century, and they have gained a lot of popularity in recent years [1]. Long before refrigerators were invented, chili peppers were used to prevent food spoilage [2]. During the Aztec era, pungent chilies were used to relieve toothaches, and centuries later researchers proved the science behind chili peppers’ analgesic properties [3]. At the core of this anti-fungal and pharmacological activity, as well as pungent taste, is a family of compounds named capsaicinoids. Capsaicin is the most well-known member of this group, and has a molecular structure consisting of an amide moiety acylated by a C10 branched-chain fatty acid. Other capsaicinoids retain the amide moiety while varying the fatty acid moiety, Figure 1.1. Analogous to capsaicinoids, there exists another compound group named capsinoids which can be found in sweet and chili peppers [4, 5]. They contain an ester bond in place of an amide bond that exists in capsaicinoids, Figure 1.1. Due to their structural differences, capsinoids have much lower pungency than capsaicinoids, however they have similar physiological activity [6]. While chili peppers’ spice pallet consist of differential synthesis of capsaicinoids and capsinoids, sweet peppers only contain capsinoids. 2 R1= Capsaicinoids Capsinoids Figure 1.1 Chemical Structure of Some of the Identified Capsaicinoids and Capsinoids Capsinoids have similar structural variety as capsaicinoids, however as they have not been heavily investigated and there are fewer compounds in the capsinoid family which have been characterized. N-Vanillyloctamide Nornorcapsaicin N-Vanillylnonamide N-Vanillyloctamide Norcapsaicin Nordihydrocapsaicin Dihydrocapsaicin Capsaicin Homodihydrocapsaicin Homocapsaicin ɪ Homocapsaicin Capsiate Nordihydrocapsiate Dihydrocapsiate δ-methylhomodihydrocapsaicin δ-methylhomocapsaicin R1= 3 Pungency has been commonly used as a technical term to describe capsaicinoid content in a sample, and also the heat sensation experienced when consuming chili peppers. Pungency can be measured via analytical techniques that report capsaicinoid concentration or via the Scoville Heat Units (SHU) organoleptic test. The latter is a less accurate assessment tool as it relies on a panel of human subjects determining the heat level experienced when tasting prepared chili samples [7]. The burning sensation experienced when coming into contact with pungent substances is caused by activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) pain receptors. The intensity of the burning sensation depends on the amount and type of capsaicinoids present in the sample. Capsaicin and dihydrocapsaicin are some of the spiciest compounds ever known to humans. Approximately 80 to 95% of the total capsaicinoid contents of chili peppers consists of capsaicin and dihydrocapsaicin [8]. Over twenty capsaicinoids have been identified in Capsicum species [9], differing by acyl chain length (typically C8 to C11), saturation, and optional branching at iso or ante-iso positions. Long-chain capsaicinoids, also referred to as long-chain N-Vanillyl-acylamides, have been identified in C. oleoresin. Their acyl chain ranges from C14 to C18 and they are only present in fully saturates or unsaturated straight-chain form [10]. The same principles appear to apply to capsinoids, however their structural variety has not been characterized to the same extent. 1.2 Medical Studies and Industrial Applications Capsaicinoids have become the target of numerous clinical studies due to their impact on the nervous system. Their pharmacological effects range from analgesia and antitumor activities, to weight loss, cardiovascular and gastrointestinal effects [11]. Beyond pharmaceuticals, capsaicinoids have found applications in nutrition, cosmetics and even in chemical repellants such as shark repellents and pepper sprays [12, 13]. Capsinoids have also shown antitumor activities, and can also affect energy metabolism in humans [14, 15]. They are less toxic than capsaicinoids, however they have largely remained marginal in research studies, possibly because they are less stable in aqueous solutions [11]. 4 Double-blind studies have demonstrated the efficacy of capsaicin in treating certain neuropathic pain conditions such as arthritis, diabetic neuropathy, and Morton's neuroma [16, 17, 18]. After application to the skin, capsaicin causes a short term increase in sensitivity, followed by a period of reduction in sensitivity [19]. In a meta-analysis of 8 randomized controlled and blinded studies on Qutenza, an FDA approved high-dose 8% capsaicin patch, about 10% more participants reported improvements in pain intensity reduction of at least 30% percent compared to control groups [20]. This is a salient result, considering that neuropathic pain is difficult to treat and only a small proportion of participants experience clinically proven benefits [20]. The mechanism of action of capsaicin is linked to the TRPV1 receptor. It has been theorized that defunctionalization and a loss of responsiveness of sensory neurons contributes to the analgesic effects of capsaicin [21]. Advantages of capsaicin treatments include longevity of pain relief, which can last several months, lower chances of drug-drug interactions, and reduced risk of systemic effects [22]. Overall, a limited number of people will benefit from capsaicin treatment and the main unanswered question is how to predict who will benefit from it [20]. Capsaicinoids have demonstrated antitumor activity in both in vivo and in vitro studies [11]. Their anticancer effects have been linked to capsaicinoids’ ability to prevent cell proliferation and induce cell apoptosis [11]. From the capsaicinoid compound group, capsaicin and dihydrocapsaicin have been most commonly referenced in research studies. The two compounds also happen to be the spiciest capsaicinoids, as well, the most abundant capsaicinoids present in chili peppers. However, anticancer activity of the two compounds is not solely linked to the stimulation of TRPV1 receptor. Many studies have demonstrated capsaicin co-treatment with TRPV1 antagonists did not protect the cells from apoptosis [23, 24, 25]. In some instances, inhibition of TRPV1 resulted in increased cytotoxic effects of capsaicin [26, 27]. In a study done by American Association for Cancer Research, it was discovered that capsaicin was able to cause prostate cancer cells to undergo apoptosis, reducing the tumor size of treated cells by one fifth [28]. The adverse side effects of capsaicin, including pain and gastric irritation, is 5 one of the hurdles preventing this compound from being employed in cancer therapy [29]. As a result, experiments have been performed to identify capsaicin analogues that do not function as an agonist for the TRPV1 receptor [29]. Anti-obesity properties of capsaicin and its mechanism of action have been extensively studied. It has been demonstrated that regular capsaicin consumption reduces appetite and increases energy expenditure [30, 31, 32]. In particular, capsaicin has been shown to inhibit lipid accumulation and modulate lipid catabolic pathway [33, 34]. Proteomic analysis of white adipose tissue in rats indicated that the regulation of thermogenesis and lipid metabolism related proteins were significantly impacted upon capsaicin treatment [35]. Meta-analysis of capsaicin and capsiate consumption in participants with BMI exceeding 25 kg/m2 showed a clear increase in energy expenditure and decrease in respiratory quotient. However, such correlation did not exist when the mean BMI of the participants was below 25 kg/m2 [36]. Overall, the analysis provided quantifiable evidence that capsaicin and capsiate could be used as a therapeutic in treating obesity. Cardiovascular benefits of capsaicin are linked to its impact on metabolism and the TRPV1 receptor. In reperfusion injuries, activation of TRPV1 results in the release of neuropeptide substance P and calcitonin gene-related peptide, both of which contribute to cardioprotection. In addition, TRPV1 activation lessens the generation of free radicals and inflammatory cytokines, and boosts the production of anti-inflammatory cytokines to limit the impact of reperfusion caused tissue injuries [37, 38]. TRPV1 agonists such as capsaicin offer cardioprotection by activating the sensory nerves and triggering a series of cardiovascular responses [39]. Pretreatment of non-diabetic mice hearts with capsaicin substantially reduced reperfusion-induced injuries. Preliminary analysis of diet and mortality data from four large international studies conducted by the American Heart Association concluded that consumption of chili pepper may reduce the relative risks of cardiovascular disease mortality by 26%, cancer mortality by 23% and all-cause mortality by 25% [40]. Several limitations were associated with the study including 6 unavailability of specific health data on individuals or other factors that may have influenced the findings as well as variability in the amount and type of chili pepper consumed among the participants. Overall there is this body of evidence illustrates potential health benefits and therapeutic use of capsaicin and chili peppers through both in vivo and population-based studies. 1.3 TRPV1 Receptor and its Relation to Pungency The transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel belonging to the TRP family. The receptor is mainly expressed in sensory neurons and is distributed across different organs including brain, heart, lungs, kidney, liver, and intestine [37]. With a molecular weight of 95 kDa in humans, TRPV1 possesses six transmembrane domains with a pore-forming region between transmembrane segments five and six [37]. The receptor mainly functions as a noxious heat (>42 °C) sensor and can be activated by low pH as well as chemical agents such as eicosanoids, capsaicinoids, protons and peptide toxins [41]. In addition to thermoregulation and pain perception, TRVP channels are involved in the maintenance of bone homeostasis and gastrointestinal functions [42]. Capsaicinoids possess three functional moieties, vanillyl, amide bond, and alkyl side chain. The vanillyl motif and the amide bond regions have been linked to the pharmacological activity on TRPV1, while the side chain impacts potency [43, 44]. This differentiation is also evident when comparing SHU ratings of different capsaicinoids and capsinoids. Capsaicin and dihydrocapsaicin only differ by the presence of a CC double bond, but have equal SHU. As the acyl chain length increases or decreases the SHU rating drops. Capsinoids which have an ester bond in place of the amide bond have substantially lower SHU. Capsaicinoids and Capsinoids can both activate the TRPV1 receptors, however pungency of capsinoids cannot be felt in mouth as the compounds cannot reach the receptors located slightly below the skin. 7 1.4 Evolution of Capsicum Species and Capsaicinoid Biosynthesis Pathway Capsicum genus belongs to the Solanaceae family, which includes economically important species such as tomato, potato, and tobacco. Members of the Solanaceae family are known to be potent alkaloid producers [45]. So far, about 35 Capsicum species have been identified [46]. The more pungent cultivars are typically referred to as chili peppers and the less piquant are known as sweet peppers. Evidence suggests that Capsicum species evolved to defend against certain vertebrates and microbial pathogens using capsaicinoids while keeping more effective seed dispersers such as birds [47, 48]. Unlike most mammals, birds’ TRPV1 receptor is insensitive towards vanilloids like capsaicin allowing them to forage on chili peppers without feeling the pain-producing effects [49]. Furthermore, capsaicinoids protect chili peppers against insects and fungi. Capsaicinoids have shown insecticidal activity towards several agricultural insects with LC50 value as low as 152.82 mg/L [50]. Researchers identified that pungency variations present in polymorphic C. chacoense is linked to the variations in the damage caused by fungal pathogens [18]. Specifically, presence of microbial attacks facilitated by insect scars created a selective pressure increasing the proportion of capsaicinoid producing plants. In the absence of microbial pressure, the non-pungent phenotype thrived [18]. The results showed how fruit chemistry can be influenced by the presence of microbial infections. Capsaicinoid synthesis in Capsicum species begins from the phenylpropanoid pathway, using phenylalanine as a precursor and leading to vanillin through multiple enzymatic reactions. A putative aminotransferase (pAMT) then converts vanillin into vanillylamine. Branched fatty acid (BCFA) synthesis relies on amino acids leucine, valine, and isoleucine to synthesize BCFA precursors isovaleryl-CoA, isobutyryl-CoA, and 2-methylbutyryl-CoA respectively. These branched acyl-CoA substrates are used to initiate fatty acid synthesis, and after multiple rounds of chain elongation, the branched-chain FAs are liberated from ACP by FatA thioesterase. For straight-chain FA synthesis, acetyl-CoA is used as a starting unit. Similar to bacterial species, plants also rely on the Type II FA synthesis pathway where the 8 synthesis reactions are carried out by individual proteins encoded by discrete genes [51]. FatA is predicted to liberate 8-methyl-nonanoic acid in Capsicum species, however there could be other thioesterases involved in the pathway for different acyl chain lengths. Acyl-CoA synthase (ACS) then converts these fatty acids into acyl-CoA esters, which are finally condensed with vanillylamine by capsaicin synthase (AT3) to produce capsaicinoids. The capsinoid pathway shares the same elements, however, due to a mutation in pAMT, the enzyme is deactivated and instead vanillin is converted to vanillyl alcohol by an uncharacterized enzyme [52]. Functional loss of pAMT in Capsicum plants has resulted in the biosynthesis of capsinoids in lieu of capsaicinoids, leading to a reduction in pungency [53, 54]. In addition, recessive or non-functional alleles have been identified that contribute to the presence or absence of pungency in the Capsicum genus. Namely, certain mutations in the promoter region and/or exon regions of AT3 and pAMT have also been linked to loss of pungency [54, 58]. Figure 1.2 shows an illustration of the synthesis pathway of capsaicin. 9 Figure 1.2 Capsaicin Synthesis Pathway Reprinted from Stewart et al. (2005). Capsaicinoids have a similar enzymatic pathway and rely on valine, leucine, and isoleucine as precursors for branched-chain FA synthesis. Capsinoids on the other hand differ by having vanillyl alcohol as a precursor instead of vanillylamine. Vanillin is converted to vanillyl alcohol by an uncharacterized enzyme. 10 The major missing element in the capsaicin synthesis pathway is the mechanism behind the appearance of the double bond at the ω-2,3 position, which is rare in plant fatty acids. In one study, the authors suggested that a desaturase enzyme could be acting on 8-methylnonanoic acid to create the product [55]. This reaction is suggested to occur while the fatty acid is fully synthesized but still bound to ACP [56]. However, in another study, it was proposed that the desaturation of the fatty acid moiety happens due to incomplete reduction of fatty acid during the elongation step [57]. To date, it is unclear which proposed pathway is responsible for the double bond. Various steps in capsaicin synthesis pathway are subject to spatial and temporal regulation. AT3, which belongs to the BAHD family and resides in the Pun1 locus, is exclusively expressed in the placenta of Capsicum fruits [58]. Expression of AT3 and accumulation of capsaicinoids begins at approximately 20 days post anthesis (dpa) and continue until 60 dpa. The distribution of capsaicinoids goes beyond placenta, and into seeds and pericarp, although in lesser amounts [59]. AT3 has been shown to catalyze both capsaicinoid and capsinoid synthesis in pungent and non-pungent pepper cultivars respectively [52]. While AT3 mutations can inhibit capsaicinoid and capsinoid biosynthesis, pAMT mutations hinder the biosynthesis of capsaicinoids only while keeping capsinoid production intact [23, 29]. Silencing AT3 results in accumulation of pathway intermediates, which reduce the expression of other genes involved in the capsaicinoid pathway [60]. Moreover, AT3 silencing has been shown to reduce capsaicin and dihydrocapsaicin contents by approximately 90% [60]. It is possible the rest of the capsaicinoids are produced by AT3 homologues and/or other acyltransferases. In transcriptomics analysis of C. frutescens, 21 acyltransferase-coding genes were detected, and they showed different expression patterns through different developmental stages, suggesting that some may play a regulatory role [61]. Thus far, AT3 is the only known enzyme to have an effect on capsaicinoids biosynthesis [62]. 1.5 Objectives of this Study The aim of this project is to assess the feasibility of using E. coli as a chassis for the biosynthesis 11 of capsaicinoids and capsinoids using vanillin and a carbon source for future exploitation in commercial bioprocesses that utilize lignin as the feedstock. E. coli was chosen due to its fast design-build-test cycle, the availability of bacterial homologues for catalyzing most pathway steps, and the ease with which E. coli can be engineered for increased fatty acid yields. Creation of a capsaicinoid pathway in E. coli requires introduction of several non-native pathway genes, Figure 1.3. Capsaicinoids and capsinoids are composed of an aryl component and an acyl-CoA component. Heterologous expression of a minimum of three enzymes in E. coli is required for straight-chain capsinoid production. A thioesterase and acyl-CoA synthase are required to make the requisite acyl-CoA intermediate. Endogenous E. coli reductases will catalyze the formation of vanillyl alcohol from exogenously-supplies vanillin. For straight-chain capsaicinoid production an additional enzyme is needed to make vanillylamine from vanillin. In the final step of the pathway, capsaicin synthase condenses vanillylamine or vanillyl alcohol with an acyl-CoA substrate to produce capsinoids or capsaicinoids. Branched-chain capsaicinoids and capsinoids require heterologous expression of two additional enzymes, one being a multi-domain enzyme complex. Furthermore, other enzymes, such as FadR transcription regulator, may need to be overexpressed to increase fatty acid titers. We broke down this pathway into four sections, corresponding to the objectives and aims of this study. Bacterial homologues were used where possible, and sometimes multiple homologues were included to evaluate their activity and yield. 1) Engineer an E. coli for straight and branched medium-chain fatty acid (FA) production (Ch.2) 2) Identify suitable acyl-CoA synthases for the activation of these acids (Ch. 3) 3) Production of vanillylamine and vanillyl alcohol from exogenously-supplied vanillin (Ch. 4) 4) Expression of capsaicin synthase in E. coli (Ch. 5) Chapter two focuses on engineering E. coli for both linear and branched medium-chain FA production. Production of branched, medium-chain FA in E. coli requires heterologous expression of two 12 non-native genes, a thioesterase and a branched alpha-keto-acid dehydrogenase (BKD). Overexpression of a thioesterase combined with the overexpression of the transcription factor FadR results in increased production of FAs. Overexpression of FadR has shown increase FA yields by 7.5 fold in ΔFadE strain [63]. We identified four thioesterase candidates to evaluate for the biosynthesis C6 to C18 straight and branched-chain FAs, they include C. viscosissima CvFatB1 which has already been expressed in E. coli and shown to hydrolyze C8 to C10 straight chain fatty acids [64], A. baylyi AcTesA’ (prime symbol refers to thioesterase with its signal sequence deleted allowing cytosolic expression) which has high homology to native E. coli TesA’ and has shown to be active towards a wide range of acyl chain lengths including medium-chain FAs with high yields [65]. Lastly we will evaluate WT E. coli thioesterase named TesA’ and then its mutant version TesA’ Y145K which has previously been reported to liberate medium-chain FAs with higher yields than WT TesA’ [66]. Adding the branched-chain α-keto acid dehydrogenase (BKD) operon and beta-ketoacyl-ACP synthase III (FabH2) from B. subtilis to the thioesterase and FadR construct will allow the synthesis of branched-chain FAs. BKD and FabH2 have been studies in the production of biofuels in E. coli [67, 68]. We have to investigate whether any of the thioesterase candidates can bind to and hydrolyze branched-chain acyl-ACPs. Chapter 3 seeks to identify a suitable Acyl-CoA synthase to create the CoA-bound medium length acyl chain. We have four candidate enzymes for this reaction that we will evaluate, the native C. annum ACS, the native E. coli FadD and its mutant version FadD V451A, as well as M. tuberculosis FadD6. The thioesterase and acyl-CoA synthase can both act as gatekeepers for the availability of acyl-CoA pool to capsaicin synthase. As a result having a number of candidates for each of the enzymes can help designing a modular system whereby switching between different thioesterase and acyl-CoA synthases it would become possible to synthesize capsaicinoids with particular acyl chain lengths. Chapter 4 focuses on the conversion of exogenously supplied vanillin to vanillylamine. For capsaicinoid production we require enzymes capable of carrying out the transaminase reaction for 13 converting vanillin to vanillylamine, and for capsinoid synthesis reductases are needed for the catalysis of vanillyl alcohol from vanillin. For vanillylamine production we will evaluate the CV2025 ω-transaminase from C. violaceum [69]. Previous in vitro experiments have shown that pAMT was only able to carry the reverse reaction in E. coli to convert vanillylamine to vanillin [70]. WT E. coli possesses reductases capable of reducing vanillin to vanillyl alcohol and we will evaluate whether it is possible to synthesize capsinoids simply by excluding the transaminase from the construct [71]. The last step of the pathway, the focus of Chapter 5, includes a condensation reaction carried out by capsaicin synthase AT3. Recombinant expression of AT3 has been challenging due to its low solubility [58]. Information obtained from a patent suggests that the SUMO tag may be able to improve solubility of the enzyme [72]. The optimum temperature for fruit set in Capsicum plants is between 16°C and 21°C [73], we will be keeping this information in mind when expressing Capsicum-based genes. These four aforementioned sections, straight and branched-chain FA production, acyl-CoA synthase reaction, transaminase, and acyl-transferase reaction will each constitute their own chapter in this report. For the engineered strain, competing pathways need to be eliminated to direct all free fatty acids towards capsaicinoid production. So far we have determined the fatty acid degradation as a potential competing pathway, which can be addressed by creating a FadD and FadE knockout strain. Figure 1.3 shows the synthesis pathway we have designed in E. coli and the potential compound libraries that can be produced using the candidate enzymes. Furthermore, we will explore the possibility for making specific molecules from either of the compound libraries. The system relies on supplying vanillin as a precursor which may not an economically viable for industrial purposes. However, there have been extensive research on methods for synthesis of vanillin in bacterial species, including using lignin as a precursor [74, 75, 76]. Successful implementation of this metabolic pathway allows for the production of variety of capsaicinoids with different acyl chain lengths, some of which naturally exist at very low abundance. 14 Figure 1.3 Proposed Capsaicinoid and Capsinoid Synthesis Pathway in E. coli Straight-chain capsaicinoid synthesis rely on the E. coli FA synthesis pathway and use acetyl-CoA as a precursor. The elongated acyl-ACP is then hydrolyzed by a thioesterase and afterwards activated by an acyl-CoA synthase. For branched-chain FA synthesis, valine, leucine, and isoleucine are converted into branched-chain α-keto acids by branched-chain amino acid transaminase. Afterwards the products are converted into their respective CoA form by a branched-chain α-keto dehydrogenase. The CoA starters enter the FA synthesis pathway via condensation with malonyl-ACP by β-ketoacyl-ACP synthase. On the other side of the pathway vanillin is catalyzed into vanillyl alcohol or vanillylamine. The last step of the reaction is carried out by capsaicin synthase which condenses vanillyl alcohol or vanillylamine with an acyl-CoA to produce capsinoids or capsaicinoids respectively. Successful expression of capsaicin synthase will allow determination of the minimum and maximum acyl chain lengths accepted by the enzyme. 15 1.6 Potential Benefits for Microbial Production of Capsinoids and Capsaicinoids Currently, capsaicinoid production methods rely on a challenging extraction and purification process. The end result is a dry weight yield of 0.01% to 1% [77]. In addition, 98% of the total capsaicinoid content of chili peppers consist of capsaicin, dihydrocapsaicin, and nordihydrocapsaicin [78]. For that reason, access to other naturally produced capsaicinoids is severely restricted. Moreover, agricultural activities are one of the major contributors to greenhouse gas emissions [79, 80]. These are some of the issues associated with current capsaicinoid production methods. Capsaicin synthase is a member of the BAHD group of enzymes which are known to possess broad substrate specificity with respect to their acyl donor and acyl acceptor. Without an engineered synthesis pathway it is not possible to determine the specificity of this enzyme for particular substrates, and it is not possible to synthesize all the potential compounds that this enzyme can produce. Capsaicin is a drug molecule and a TRPV1 agonist. There are ongoing TRPV1 modulator drug design programs to develop TRPV1 antagonists with reduced side effects including accidental burns and hyperthermia [81]. Having a modular capsaicinoid and capsinoid production system could allowed tailored production of TRPV1 agonists with different affinity levels, as well be a step towards customizing ligand binding to receptors for manufacturing drug molecules. Capsinoids and capsaicinoids have demonstrated utility as preservatives, flavorings and therapeutics. While chili plants suffice for some applications, microbial production may enable enhanced yields and greater control over the type of capsaicinoids and capsinoids produced. Moreover, by expanding the repertoire of economically-relevant molecules produced through microbial fermentation, alternative uses for waste streams such as lignocellulosic biomass would become possible. To this end, this thesis explores the development of a microbial strain for capsinoid and capsaicinoid production. 16 Chapter 2 Biosynthesis of Medium Straight and Branch-Chain Fatty Acids 2.1 Introduction Free fatty acids (FFAs) are an essential element of the capsaicinoid and capsinoid (CC) biosynthesis pathway and are one of the determining elements of the CC lipid chain-length. Biosynthesis of FFAs in microorganisms has been pursued as an avenue towards the production of biofuels, biomaterials, biochemicals as well to replace petroleum feedstock. FFAs can be converted into alkanes, alkenes, or fatty alcohols. Branched-chain fatty acids in particular are suitable for advanced biofuels such as diesel and jet fuel [82]. Properties such as degree of saturation, chain length and branching are directly correlated with energy content, melting point, and volatility of the compound [83]. Hence, researchers have dedicated their efforts to tune the aforementioned properties. Furthermore, medium-chain FFA have garnered some attention due to their applications for producing medium-chain oleochemicals [66]. This has resulted in publications targeting those compounds, and since capsaicinoids also use medium-chain FFAs, we will leverage this information in our research. 2.1.1 Straight-chain Fatty Acid Synthesis and Regulation84 E. coli relies on the type-II FA synthesis enzymes for de novo synthesis of FAs. Type-II FA synthesis is mainly present in prokaryotes and in the plastids of plants, and in this system each reaction is catalyzed by discrete enzymes [85]. The starting units for FA synthesis in E. coli are acetyl-CoA and malonyl-ACP (acyl carrier protein), which are condensed by FabH to yield acetoacetyl-ACP, Figure 2.1. Odd-chain FAs can be produced using propionyl-CoA in place of acetyl-CoA in the initial condensation reaction. The product is then reduced by FabG and dehydrated to 2-butenoyl-ACP by FabZ or FabA. The two enzymes have broad overlapping chain length specificities [86]. However, FabA can also isomerize trans-2-decenoyl-ACP into cis-3-decenoyl-ACP, which becomes the main precursor for unsaturated FA synthesis [86]. Subsequent to the dehydration reaction, FabI catalyzes the last step by reducing 2-butenoyl-ACP to butyryl-ACP. For the second and subsequent FA elongation cycles, FabB and FabF carry 17 the condensation of malonyl-ACP with fatty acyl-ACP. There is some variation between the substrate specificity of FabB and FabF, one of them being the ability of FabB to condense certain unsaturated fatty acyl-ACPs. The condensation, reduction, and dehydration cycles continue until TesA liberates the FA from acyl-ACP to create FFA. The specificity of the thioesterase plays a vital role in the FFA pool. 2.1.2 Thioesterase Acyl Chain Length Selectivity and Engineering Thioesterases (TEs) consist of a large enzyme group responsible for the hydrolysis of the thioester bond between a carbonyl group and a sulfur atom. Thioester bonds are typically present in acyl-ACPs, acyl-CoAs, glutathiones, and on acylated proteins. TEs are classified into 23 families, with acyl-ACP TEs included in family 3 (TE3) [87]. TE3 have a flavodoxin-like fold, are mainly found in bacteria, and have acyl-CoA hydrolase, arylesterase, and lysophospholipase activities. The proposed catalytic Figure 2.1 Straight-chain FA Synthesis Pathway Reprinted from Luo et al. (2016) with modifications. FA elongation cycle includes four steps, condensation by FabH, reduction by FabG, dehydration by FabZ or FabA, and then reduction by FabI. FabA and FabZ have overlapping substrate specificities, however only FabA can synthesize unsaturated FAs by isomerizing trans-2-decenoyl-ACP into cis-3-decenoyl-ACP. The product is then condensed by FabB. The second cycle begins by the condensation of butyryl-ACP with malonyl-ACP catalyzed by FabF or FabB. FabB can condense certain unsaturated fatty acyl-ACPs while FabF can only accept fully saturated FAs as substrate. FAs are synthesized in two-carbon increments. After multiple elongation cycles the fatty acyl-ACP is liberated by TesA to create FFAs. 18 residues of this family are the Ser10, Asp154, and His157 triad [87]. Members of this family typically have TesA as part of their nomenclature. The mechanism underlying thioesterase alkyl chain length specificity is not fully understood. Ziesack et al. (2018) conducted an in-depth study of TE/acyl-ACP interaction by creating chimeras of C. palustris CpFatB1 (C8 specificity, lower activity) and CpFatB2 (C14 specificity, higher activity) performing site-directed mutagenesis, and in silico modeling [88]. The authors concluded that there are three possible contributing factors to TE substrate specificity: 1) Sheltering of the fatty acid inside the ACP pocket and protection from the aqueous phase; 2) presence of a positive patch on the TE resulting in appropriate biding to acyl-ACP and extrusion of the acyl chain from ACP pocket; 3) and appropriate binding of the thioesterase and the acyl chain within the catalytic region. Identifying heterologous thioesterases that are simultaneously highly active and highly selective towards a particular chain length have been challenging. Considerable effort has already been invested towards engineering thioesterases with improved activity and substrate selectivity. However, these have not always been successful: U. californica FatB crystal structure was determined by Feng et al. (2017) and C12 specificity of the enzyme was rationally redesigned towards C14 through mutations in the binding pocket. However, the overall FA yields experienced a decrease (98 mg/L vs 78 mg/L) [89]. With the advent of computational tools, the wild type E. coli thioesterase, TesA’ (The prime symbol denotes a TesA variant with its leader sequence removed for cytosolic expression), was successfully engineered with higher specificity towards medium-chain FAs. Using structure-guided mutagenesis and optimization algorithms Grisewood et al. (2017) tuned substrate binding of TesA’ and simultaneously increase its kcat values [66]. After four rounds of mutagenesis, the top mutants had FA profile composing of 50% C8 or 49% C10. Similarly, Hernández Lozada et al. (2018) were able to isolate a C. palustris FatB1 mutagenized thioesterase variant with >90% specificity towards C8:0-ACP, and a yield of 1.7 g/L, after optimizing the 19 expression levels. The native enzyme expressed under the same conditions had 15 times lower yield [90]. This work was made possible by developing a genetic selection circuit based on the lipoic acid requirement of E. coli for protein lipoylation and growth. Compared to other thioesterase engineering attempts, this work was the most successful with respect to fine tuning thioesterase substrate specificity while increasing overall yields. Implementation of selective pressure and elimination of the need to individually analyze the FFA composition of each variant made a high throughput platform for thioesterase engineering. 2.1.3 Fatty Acid Chain Elongation Regulation beyond the TE Besides the TE, a number of other enzymes and regulatory networks contribute to chain length regulation of FA biosynthesis. Availability of acyl-CoA starter units, and the specificity of FabH for particular starters have a direct impact on the FA profile. E. coli 3-ketoacyl-ACP synthase, FabH, has a strong preference towards acetyl-CoA and to a lesser extent towards propionyl-CoA [91]. While E. coli FabH in incapable of condensing branched-chain acyl-CoAs with malonyl-CoA, FabHs from B. subtilis, L. monocytogenes, and S. aureus primarily use branched-chain acyl-CoAs [91]. FabB and FabF are the other ketoacyl synthases, but with specificity towards longer acyl-ACPs (C4-C14) [92]. Higher activity rate of FabB and FabF relative to FabH prevents the accumulation of short and medium chain acyl-ACPs [93]. Overexpression of FabH leads to increased C14 and C16 and reduced C18:1 production in E. coli while deletion of FabH improves the production of C18 species [94]. Altering the E. coli lipid pool has also been attempted via acyl carrier protein (ACP) engineering. S. elongatus ACP mutants, heterologously expressed in E. coli, were generated with reduced acyl chain pocket size resulting in 8-fold increase in dodecanoic acid production (~20 mg/L vs. ~160 mg/L) when C12-specific TE from C palustris was coexpressed with the mutant ACP. As a control non-mutant ACP was coexpressed with the TE [95]. The importance of the acyl-ACP in FA profile was also demonstrated by Feng et al. (2018) C. hookeriana ChFatB2 has a strong preference towards C10:0-ACP when expressed in 20 E. coli and 84.9% of accumulated FAs are C8:0. However, when using synthetic acyl-ACP pool in vitro to measure ChFatB2 substrate preference, C10:0-ACP activity was observed to be twice as high as C8:0-ACP [96]. It was hypothesized that the activity of the E. coli ketoacyl ACP synthases, FabB and FabF and the availability of the endogenous acyl-ACP pool had resulted in such discrepancy between in vivo and in vitro substrate preference. These results demonstrate how the interactions between TE and ACP can shift the FA pool of the host and increase FFA yields. In addition to the TE, a number of other endogenous enzymes namely FabH, FabB, and FabH can impact the FA pool of the host. Furthermore, the FA extraction and analysis methods have an impact on the results and the observed FA profile. Such elements must be taken into consideration when comparing results across different publications to determine candidate genes for the capsaicinoid pathway. 2.1.4 Fatty Acid Production Titers Recent attempts at producing FFA have reached titers as high as 21.6 g/L (0.636 g/L h productivity) in fed-batch fermentation using CRISPR interference strategy and performing omics analyses to identify targets for consecutive rounds of CRISPR interference [97]. This was achieved through multiple rounds of transcription optimization which initiated with the repression of 108 genes belonging to upstream carbon flux diversion, downstream carbon flux diversion, amino acid metabolism, β-oxidation and transcription factors. In consecutive rounds proteome and transcriptome analyses were performed to identify candidate gene targets for repression or overexpression [97]. Other attempts at producing fatty acids had achieved a production yield of 8.6 g/L (0.124 g/L h productivity) through deletion of β-oxidation pathway acyl-CoA synthase (fadD), boosting malonyl-CoA flux by overexpressing the four subunit acetyl-CoA carboxylase (accaABCD), and lastly by optimizing the expression of modules related to acetyl-CoA formation, acetyl-CoA activation, and malonyl-ACP consumption [98]. Overexpression of a thioesterase is generally the key component for increasing titers, but also 21 overexpressing and FadR in a ΔFadE or ΔFadD strain allows yields close to 5 g/L in culture flasks [63]. Figure 2.2 illustrates how the β-oxidation pathway as well as the global regulator FadR impact FA synthesis. FadR activates the unsaturated FA synthesis pathway as well represses the FA degradation pathway genes. Meanwhile β-oxidation pathway catabolizes the FFAs two carbon units at a time eventually leading to the synthesis of acetyl-CoA. Previous studies had shown that the β-oxidation knockout did not have any noticeable impact on C12 FA production [95]. This suggests that β-oxidation pathway does not have significant impact on short and medium-chain FA yield. Figure 2.2 Regulation of Fatty Acid Synthesis and Degradation Reprinted from Janßen et al. (2014) with modifications. Global transcription regulator FadR activates FA synthesis genes by binding to the promoter region. Similarly, promoter region of FA degradation genes have FadR binding site. In its default state FadR is bound to the DNA. In the presence of long chain acyl-CoA, FadR goes under conformational change resulting in its dissociation from its operator sites. [99] 22 2.1.5 Branched-chain Fatty Acid Synthesis Branched-chain fatty acid (BCFA) production in E. coli has not been investigated as extensively as straight-chain fatty acid (SCFA). The main precursor units for BCFA synthesis are branched-chain alpha keto acids which are synthesized from valine, leucine, and isoleucine by the endogenous Ilve enzyme. This will allow production of odd and even chain iso-fatty acids, as well as odd chain ante-iso-fatty acids, e.g. 11-methyl-dodecanoic acid (C13:0 iso), 12-methyl-tridecanoic acid (C14:0 iso), and 14-methyl-hexadecanoic acid (C17:0 ante-iso). Heterologous branched-chain α-keto acid dehydrogenase complex (BKD) then coverts branched-chain α-ketoacids into branched-chain α-ketoacyl-CoA starters (BCCSs), and then a heterologous β-ketoacyl-(acyl-carrier-protein) synthase (FabH) condenses malonyl-ACP with the BCCSs. A number of bacterial species carry the enzymes essential for BCFA production, including B. subtilis, S. aureus, and L. monocytogenes. In an initial attempt at producing BCFA in E. coli, Haushalter et al. (2014) were able to achieve an approximate yield of 180 mg/L by overexpressing E. coli TesA’ and FadR plus B. subtilis FabH2 and BKD in a ∆fadE E. coli strain [67]. In separate work, replacing the E. coli FabH with S. aureus FabH made it possible to exert better control over iso vs ante-iso BCFA composition and increase BCFA proportions [91]. More recent attempts at producing BCFA managed to achieve titers as high as 276 mg/L by overexpressing E. coli lipoyl ligase A (LplA) combined with lipoic acid supplement [100]. BKD overexpression depletes the lipoylation capability of E. coli resulting in an overall reduced activity of lipoylation-dependent enzymes in the cell. Some of these, such as pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, are essential for cell growth [100]. BCFA production in non-E. coli-based systems have also been attempted. The highest reported BCFA titer in bacterial cells to-date, 354.1 mg/L, has been achieved in an engineered S. coelicolor host [101]. 23 2.1.6 Thioesterase Candidates Since several bacterial thioesterases have been characterized and engineered, we selected candidate capsaicinoid pathway thioesterases using the following criteria:  Proven experimental data demonstrating the ability of the thioesterase to target medium and long-chain FAs  Prior research regarding cleavage of branched-chain FAs  Successful expression of the enzyme in E. coli in prior studies.  Reported yield of the thioesterase E. coli TesA and met all the above criteria except that the enzyme could only hydrolyze long-chain FAs. Throughout our literature review we only observed WT E. coli TesA’ being co-expressed with FabH2 and BKD for BCFA production [67, 102]. TesA belongs to the TE3 family and we postulated that other thioesterases in this family could hydrolyze branched-chain acyl-ACPs as well. Therefore, we began searching for TE3 thioesterases with medium-chain acyl-ACP preference. Through literature review we came across A. bayly TesA’ (AcTesA’) which had previously been expressed in E. coli and had a profile consisting of C6 to C18 FFAs [65]. This enzyme belongs to the TE3 family and its catalytic triad consist of Ser10, Asp158 His161 [65]. Overexpression of AcTesA’ under T7 promoter in LB supplemented with glycerol resulted in 222.9 mg/L FFA production in Bl21(DE3) E. coli strain. We will keep this information in consideration and compare our results. We were also searching for a TE that has a narrow substrate specificity with the aim of producing capsaicinoids with a particular acyl-chain length. C. viscosissima CvFatB1 appeared as a suitable option. About 80% of the total FFA produced by this thioesterase consists of octanoic and decanoic acid, the overall yield was approximately 90 mg/L when E. coli K27 ΔFadD was grown in M9 media supplemented with glucose [103]. Lastly, we picked the WT E. coli TesA’ and a mutant version TesA’ Y145K. These two thioesterases have shown approximate yields of 420 mg/L and 860 mg/L respectively when expressed under pBAD promoter in E. coli K12 ΔFadD grown in LB 24 supplemented with glycerol [66]. In addition to higher yields, TesA’ Y145K has improved specificity towards medium-chain FAs with 25% of the FFAs produced being C8 to C10. In comparison 7% of TesA’ FFAs consist of C8 to C10. Considering that C14 to C18 capsaicinoids had previously been detected in chili peppers [10], we hypothesized that the WT E. coli TesA’ could supplement the required FA for long-chain CC synthesis. It remained to be seen which one of our chosen TEs can hydrolyze BCFAs. We have included a summary of our thioesterase candidates and their relevant features below:  E. coli TesA’ (C12 to C18 FA profile, proven to cleave BCFAs)  E. coli TesA’ Y145K (C6 to C16 FA profile, twice the yield of TesA’, no information regarding BCFA production)  A. bayly AcTesA (C6 to C18 FA profile, same family as TesA’, no information regarding BCFA production)  C. viscosissima CvFatB1 (C8 to C10 FA profile, plant TE, no information regarding BCFA production) 2.2 Materials and Methods 2.2.1 Expression Vectors As there could be complications associated with the expression of polycistronic mRNA, we decided in advance to use the pET28a vector and add an additional set of Lac operator, T7 promoter, and T7 terminator. This could potentially exert better control over the ratios of expressed genes, as well reduce the length of the transcribed mRNA. There is a negative correlation between mRNA stability and CDS length, although weak [104]. In addition, having a polypromoter system has been recommended over using polycistronic systems [105]. Lastly, having two MCSs simplifies insertions of multiple fragments. If additional genes need to be inserted in the construct later on, the addition of an extra MCS simplifies the process. Initially pET-28 a(+) was digested at XbaI and BlpI sites. Afterwards using primers MCS1_F and MCS1_R as well as MCS2_F and MCS2_R, listed in supplementary Table 1, the two multiple cloning sites from pACYCDuet-1 plasmid were amplified. Assembly of the amplicons with the linearized pET-28a(+) 25 was performed using Gibson Assembly (GA). This vector which was named pET28D was used for all the experiments in this research, Figure 2.3. We opted for not using the pACYCDuet-1 due to the fact that the vector has two promoters and one terminator making the second promoter region transcribed twice. This could hinder the ability to control gene expression levels through RBS library design if one of the MCSs is being transcribed twice. Figure 2.3 Visual Depiction of pET28D Vector This vector was created by amplifying the two MCSs from pACYCDuet-1 and inserting them into linearized pET-28a(+). 2.2.2 Designing and Cloning Constructs The cloning work to create SCFA constructs was done in the following order: Double-stranded DNA encoding CvFatB1 and AcTesA’ were codon optimized and synthesized by IDT and Genewiz respectively. DenovoDNA RBS calculator was employed to ensure RBS translation rates were relatively consistent and to ensure translation was occurring adequately [106]. Based on the information from RBS calculator, we designed primers for our constructs. AcTesA’ was amplified using SF211 AcTesA’_F+pET28D/SF211 AcTesA’_R+pET28D the suquences of which can be found in Supplementary Table 1. FadR was amplified from E. coli BL21 (DE3) genomic DNA using SF211 FadR_R+pET28D/SF211 FadR_F+pET28D. pET28D vector was linearized at BglI/PshAI/XhoI/AvrII sites. SF211 construct was created using CPEC [107] and was subsequently transformed into Trans1-T1 competent cells. Clones were screened using colony PCR primers listed in Supplementary Table 2. After sequence verification SF211 construct was digested at XhoI/PacI sites. TesA’ was amplified from E. coli genomic DNA with SF111 TesA’_F+pET28D/SF 111 TesA’_R+pET28D. TesA’ Y145K point mutation was created using PCR mutagenesis with ecTesA_F (Y145K)/SF111 TesA’_R+p28D and ecTesA_R (Y145K)/SF111 TesA’_F+p28D primers. PCR overlap extension was subsequently employed to assemble to two fragments to create TesA’ (Y145K). CvFatB1 was amplified with SF311 FatB1_F+pET28D/SF311 FatB1_R+pET28D. The three 26 amplified fragments were separately inserted into the linearized SF211 vector using GA and Trans1-T1 competent cells were transformed afterwards to create SF111, SF112, and SF311 constructs, Figure 2.2 a). The following steps were iterated to create the BCFA constructs. SF211 constructs was linearized using XhoI/PacI restriction enzymes. AcTesA’ was amplified with SF211 AcTesA’_F+pET28D/BF211 AcTesA’_R+pET28D. FabH2 and BKD were amplified from B. Subtilis 168 genomic DNA using BF111 FabH2_F+TE_R/BF111 FabH2_R+BKD_F and BF111 BKD_F+FabH2_R/BF111 BKD_R+pET28D primers respectively. We then attempted to create the BF211 construct using four-fragment GA. After multiple rounds of failed attempts we decided to use the Twin-Primer Assembly (TPA) method and turned out to be successful [108].Primers used to create the fragments for TPA are listed in Supplementary Table 3. To create the rest of the BCFA constructs, BF211 was initially linearized at XhoI/PacI sites. Thioesterases were then amplified using their respective primers: TesA’ and TesA Y145K were amplified using SF111 TesA’_F+pET28D/BF111 TesA’_R+pET28D, and CvFatB1 was amplified using SF311 FatB1_F+pET28D/BF311 FatB1_R+pET28D. Two-fragment GA was performed to create the BF111, BF112, and BF311 constructs, Figure 2.2 b). All the SCFA and BCFA constructs were transformed into BL21(DE3) for expression. Supplementary Table 4 includes a list of the plasmids created in this work and their respective targeted outcomes. The sequence of the expressed genes and the GenBank accession numbers are listed in Supplementary Table 5. 27 Figure 2.4 Visual Depiction of Fatty Acid Constructs XhoI and PacI restriction sites were placed spanning the TE to simplify switching and testing of new TEs using GA. 2.2.3 Fatty Acid Production and Analysis Plasmids were transformed into E. coli BL21 (DE3) and plated on LB-agar (Miller) supplied with 50 μg/mL Kan. Expression strains are listed in Supplementary Table 4. Colonies were picked from agar plates and grown in LB Broth (Miller). 20uL of overnight cultures were then using to inoculate 2 mL LB Broth (Miller) with 1% glycerol. After reaching OD ~ 0.5 cells were induced with 1mM IPTG and placed at 37 °C for 48 hours. For fatty acid extraction, 1.5 mL culture was centrifuged at 21,000 g for 1 minute to pellet the cells, 1.2 mL of the supernatant was then transferred to Sarstedt 2 mL centrifuge tubes. Fatty acid extraction was performed as explained by Torella et al. (2013) with some modifications. 150 µL 10% NaCl, 150 µL glacial acetic acid, and 400uL ethyl acetate were added. 30 μM (3.9 mg/L) of heptanoic acid was added as internal standard. The solution was then vortexed for 20 seconds and afterwards centrifuged at 21,000 g for 10 minutes. 200 µL of the fatty acid extract was placed in glass inserts and then inside 1.5 mL vials. Analysis was done on a Perkin Elmer Clarus 680 GC and SQT 8 MS using an Agilent HP-5ms column. Injection volume was set to 4 µL and injection mode was set to 1:20 split. Initial a) b) 28 oven temperature was set to 50°C for 1 min. The method ramped from 50 to 270°C at 10°C per minute. Retention times, in comparison to a ladder of linear medium-chain fatty acids, as well as NIST library predictions were used to determine compound identities. Approximate yield of the fatty acid constructs was calculated by normalizing the peak areas against the heptanoic acid internal standard and then multiplying the obtained values by the concentration of heptanoic acid. Detailed breakdown of the calculations are shown in Supplementary Table 6. GC-MS chromatograms can be seen in Supplementary Figure 1. 2.3 Results Results from SF211 (TE: AcTesA’) demonstrated production of FAs similar to previous findings, Figure 2.5 a). The approximate yield of the SF211 construct was 125mg/L, which is less than the yield reported by Zheng et al. (2012), 222.9 mg/L. We also noticed the absence of long-chain FAs in our chromatograms, which is contradictory to previously published results, Figure 2.5 c). CvFatB1 was one of the first TEs that was tested, and we observed C8 and C10 FA peaks in our chromatograms. Due the absence of proper negative control and internal standards, these results require further verification to be conclusive. GC-MS results for strain BF211 (TE: AcTesA’) also showed clear shift in the FA profile, providing strong evidence that BCFAs within C6-C12 range are being synthesized, Figure 2.5 b). The approximate yield of the branched-chain AcTesA’ construct was 45 mg/L. Due to absence of internal standards, we were unable to identify the BCFAs with high accuracy, and had to rely on retention times as well as NIST library predictions. However, through literature review we learned that the elution of fatty acids in GC analysis happens in the following order: 1) iso-fatty acid 2) ante-iso-fatty acid 3) straight-chain fatty acid (e.g. iso C8:0, ante-iso C8:0, and straight-chain C8:0). Furthermore, branched alkanes typically fragment at the position where the branch is located producing smaller molecular ions. On this basis we predicted the FA profile of our BF211 construct. In the BF211 construct we often observed peaks corresponding to 29 2-methylbutanoic acid, which did not exist in uninduced samples. This observation needs to be investigated further to confirm that the predicted FA by the NIST library is indeed correct. Due to time constraints, we were unable to fully test TesA’, TesA’ Y145K constructs. 30 Figure 2.5 FA Profile of AcTesA Constructs and Comparison with Prior Publications a) and b) FA profile of straight-chain vs branched-chain AcTesA construct. A clear shift in the FA profile was observed when AcTesA’ was paired with FabH2 and BKD, suggesting that short and medium BCFAs are being synthesized. Peaks have been normalized with 30 μM (3.9 mg/L) heptanoic acid internal standard. c) Reprinted from Zheng et al. (2012). AcTesA SCFA profile. Contrary to prior publications our SCFA and BCFA constructs did not produce long-chain FAs. d) Reprinted from Haushalter et al. (2014). TesA SCFA and BCFA profile. When TesA’ was paired with FabH2 and BKD, the produced BCFAs generally constituted of lower acyl-chain lengths compared to the standard SCFA profile of TesA’. AcTesA SCFA Profile as Reported by Zheng et al. c) TesA SCFA and BCFA Profile as Reported by Haushalter et al. d) a) b) 31 2.4 Discussion We demonstrated, for the first time, the possibility of synthesizing medium BCFA using the AcTesA’ thioesterase. We observed a variety of iso and ante-iso FAs in the C6 to C12 range with higher peaks observed near octanoic acid. These results are in agreement the SCFA profile of AcTesA’ showing a high preference for octanoic acid. However the presence of 2-methylbutanoic in such high amount was unexpected. A number of hypothesis can explain this finding. The presence of the branched methyl group could assist in better positioning the thiol group near the TE active site causing cleavage of the FA. Another potential element is the inability of ACP’s pocket to accommodate BCFAs. Under such circumstances the acyl chain will remain exposed throughout the elongation steps and as soon as the acyl chain length reaches the minimum cleavage threshold for AcTesA, the FA is hydrolyzed. For comparison, when the E. coli TesA was paired with FabH2 and BKD by Haushalter et al. (2014), the BCFA profile consisted of C13 to C16 in relatively similar quantities Figure 2.5 d). While overexpressed E. coli TesA has a SCFA profile consists of 40% C16 to C18 FAs, when paired with FabH2 and BKD the BCFA profile is mainly distributed in the C13 to C15 region. Unlike AcTesA, TesA did not produce a particular BCFA in such high percentage. We noticed that the 2-methylbutanoic acid peak eluted right after the solvent front meaning that if 3-methylbutanoic acid was also being synthesized it may not have produced a peak on the chromatogram, Supplementary Figure 1 c). The iso form of the FAs elute earlier than the ante-iso form and therefore our GC protocol needs to be adjusted for future experiments to ensure that short-chain FAs are not being eluted before the solvent front. A potential solution would be decreasing the initial hold temperature which will reduce compound elution times. The solvent front could also be adjusted taking into consideration the elution time of the solvent which was ethyl acetate in our case. The presence of C6 to C12 in our straight-chain AcTesA’ construct was in agreement with previous findings. However, we observed a lot of variability in the FA pool of the straight and branched-chain 32 AcTesA’ constructs. Particularly, presence of C12 to C18 FAs varied from batch to batch. Previous reports have shown presence of hexadecanoic acid in WT E. coli FA extracts. In our experiments induced and uninduced cultures contained barely any long-chain FAs. It is possible that extracting FAs by pelleting the cells and using only the culture media resulted in the absence of long-chain FAs in the chromatograms. In contrast to medium and short-chain FAs, long-chain FAs do not freely diffuse across E. coli membrane. They can only uptaken by the transporter protein FadL [109]. This results in the majority of long-chain FAs remaining inside the cell. The other contributing factor to the absence of long-chain FAs could have been the FadR transcription regulator. FadR represses the β-oxidation regulon genes in its default state, and upon binding to long-chain acyl-CoAs it detaches from its operator sites resulting in the expression β-oxidation pathway genes including FadD and FadE. Different FadR variants exist in different E. coli strains and their specificity of acyl-CoA chain length varies. The variability that we observed in our long-chain FA chromatogram peaks could also have been due to deactivation of FadR transcription repressor in the presence of long-chain FA and as a result, activation of β-oxidation pathway, which utilizes long-chain FAs. Lack of derivatization could have also contributed to the low reproducibility of our results. Short-chain FAs including C5 and C6 are more volatile and can evaporate during the extraction procedure if they are not derivatized. Some of the FA peaks were overlapping other compounds, e.g. hexanoic acid and glycerol, making accurate estimation for those compounds challenging. Furthermore, using normalized results will only provide a rough estimate of the fatty acid yields. At the same concentration, longer chain FAs produce larger peak areas relative to shorter chain FAs, hence using heptanoic acid to predict the concentration of tetradecanoic acid or longer chain FAs will not produce highly accurate results. 33 As mentioned previously, it has been proposed that the formation of the double bond in capsaicin is due to incomplete activity of enoylreductase during fatty acid synthesis [57]. In E. coli the same mechanism results in the production of unsaturated fatty acids. Hence, it may be possible to utilize the unsaturated fatty acid pathway in E. coli to produce unsaturated capsaicinoids such as capsaicin. We observed C10:1 and C12:1 monounsaturated FAs in the AcTesA’ overexpressing strain. Therefore, this pathway could potentially be used to create precursors for unsaturated CC biosynthesis. 2.5 Future Work Our TE choices were certainly not comprehensive, there are numerous other thioesterases that can be tested for BCFA production. AcTesA can synthesize branched C6 FAs in relatively high amounts. There could be TEs with preference for other BCFA, e.g. C10 or C12. We specifically designed our BCFA plasmids for easy TE substitution. By linearizing the vector using XhoI and PacI restriction enzymes, the TE is removed and new TE can inserted using GA. Later on during the project, we realized that there were better options, specifically using CpFatB1.2-M4-287 [90] would have been a better choice over CvFatB1. The engineered CpFatB1 has a much higher FA yield, but the specificity is as narrow as CvFatB1. A highly selective thioesterase is suitable when production of a particular compound is sought. On the other hand if the objective is to produce a particular range of compounds, e.g. C6 to C12 capsaicinoids a thioesterase with broad substrate specificity such as AcTesA’ may be a better option. The availability of a wide range of thioesterases provide the opportunity to synthesize a variety of capsaicinoids with unique chain lengths and branch configurations. As discussed in section 2.1.5 deletion of the E. coli FabH in combination with overexpressing the S. aureus FabH would allow better control over iso vs ante-iso FA formation when supplying individual branched-chain alpha keto acids. This modification might be useful when attempting production of particular compounds within the capsaicinoid and capsinoid libraries. Furthermore, overexpressing E. coli lipoyl ligase A (LplA) combined with lipoic acid supplement could increase BCFA titers by 50% based 34 on previously published results [100]. Overall, BCFA regulation is more challenging as it is depended on the specificity of the thioesterase, β-ketoacyl-(acyl-carrier-protein) synthase, and the availability of branched-chain α-ketoacyl-CoA starters. 2.6 Conclusion We were able to demonstrate the synthesis of medium BCFAs using AcTesA’. Presence of BCFAs were prominent in the C7 to C11 region, which corresponds to the high octanoic acid production of the straight-chain AcTesA construct. This illustrates the preference of AcTesA for acyl-ACPs that are close to C8 in length. As shown in Figure 1.1 the majority of identified capsaicinoids and capsinoids have a lipid chain length ranging from C8 to C11. SCFA and BCFA profile of AcTesA corresponds well to the acyl chain length of the naturally existing CCs. The presence of large amounts of 2-methylbutanoic acid was unexpected, and further investigation is needed to determine whether AcTesA can synthesize other short BCFAs. To our knowledge pairing of AcTesA with B. subtilis FabH2 and BKD for the production of BCFAs has not been attempted previously. GC chromatograms show a clear shift in FA profile corresponding the BCFAs in iso and ante-iso form. Overall, our results demonstrate that AcTesA might be a suitable candidate for CC synthesis. Upon collection of further data, and improvement of FA titers, it would be possible to obtain publishable results. 35 Chapter 3 Identifying a Suitable Acyl-CoA Synthase 3.1 Introduction Prior to the condensation reaction by capsaicin synthase, the FFAs must be activated to their respective CoA form. This reaction is carried out by fatty acyl-CoA synthase (ACS). Acetyl-CoA synthetases have also been referred to as ACSs, however they constitute a different enzyme group and should not be confused with acyl-CoA synthases (also referred to as ACSs in other works). Acyl-CoA synthases with fatty acid substrates are sometimes called fatty acid-CoA synthetases (FACSs). Activation into acyl-CoA esters direct FFAs towards certain metabolic pathways, including β-oxidation, polyketide synthesis, and synthesis of complex lipids synthesis [110, 111]. ACSs also play important roles in enzyme regulation, posttranslational protein acylation, and transcription [112]. The particular role of ACSs across species vary, in E. coli they are only responsible for activating the FAs for degradation. Similar to thioesterases, several acyl-CoA synthases have also been characterized for potential use in microbial biofuels production [113, 114, 123]. These enzymes provide a resource from which ACS candidate selection can be made. The activation of FAs by ACS happens as a two-step reaction. Initially, the acyl-adenylate intermediate is formed with the release of pyrophosphate, and AMP is subsequently exchanged with CoA through nucleophilic substitution [116]. Acyl-CoA synthases belong to the adenylate-forming enzyme superfamily which includes other sub-families such as non-ribosomal peptide synthetases and luciferase oxidoreductases [115]. The ATP/AMP binding signature motifs (GYGLTE and YTGGTTGVAKGA) can commonly be found in this enzyme superfamily, Figure 3.1 a) and b) [116]. For the synthesis of CC, we sought enzymes capable of activating C6 to C18 straight or branched-chain FAs into their respective CoA form. 36 3.1.1 Acyl-CoA Synthase Chain Length Selectivity and Engineering Two motifs have been implicated in ACS substrate selectivity. The FACS motif, a signature motif with a 25-amino acid consensus sequence, DGWLHTGDIGXWXPXGXLKIIDRKK, is commonly found in the fatty acyl-CoA synthase subfamily, Figure 3.1 c) [117]. The M domain, which partially overlaps with the FACS motif, has also been implicated in ACS chain-length selectivity [120]. In an ACS engineering attempt done by Ford et al. (2015), two of seven variants which increased FadD specificity towards octanoic and hexanoic acid, had mutation located in the M-domain. This suggested that ACS engineering with enhanced specificity towards medium-chain FA may be possible by comparing the sequences across the M domain, Figure 3.1 d). a) b) c) d) Figure 3.1 Four Conserved Protein Regions of Acyl-CoA Synthases E. coli FadD, E. coli FadK, P. putida PP_0763, Streptomyces sp. RevS, M. tuberculosis FadD6, M. avium Mig, C. annum ACS. a) and b) ATP-AMP signature motifs found in adenylate-forming enzymes including acyl-CoA synthases. c) Fatty acid-CoA synthetase (FACS) consensus sequence. d) M-domain of ACSs, FadD F447S and FadD V451A, marked by the red rectangles, demonstrated increased activity on hexanoic and octanoic acid compared to WT FadD. 37 In contrast to thioesterase engineering, very few publications have attempted ACS engineering. There are multiple channels in the ACS proteins with multiple substrate-binding and product exiting steps, and this adds to the complexity of engineering these proteins. Ford et al. (2015) enhanced E. coli ACS FadD activity on medium-chain FAs using error prone PCR and growth-based selection strategy [113]. It was hypothesized that the mutations facilitate AMP exit from the active site by opening the corresponding pocket [113]. The top variants had a Vmax value more than twice the WT variant on octanoic acid. Because of these complexities, there has been greater focus on screening and characterization of existing ACSs in lieu of engineering substrate specificity. 3.1.2 Evaluated ACSs in E. coli and Challenges in ACS Comparisons E. coli possesses two endogenous ACSs, FadD and FadK, which are involved in the aerobic and anaerobic β-oxidation pathway respectively [118, 119]. While FadK can activate FFAs as short as C4, the shortest acyl-chain, FadD can activate is C8 [119]. However, the absolute activity of FadK is substantially lower than FadD. Enzyme activity assays revealed that FadD can activate decanoic acid at a rate of ~90 nmol/min per mg of protein when supplied with 1 mM of fatty acid, on the other hand FadK catalyzes the reaction at ~0.6 nmol/min per mg of protein. M. avium macrophage-induced gene mig encodes a medium-chain ACS and its FA activation profile has been studied in E. coli [120]. Mig has preference for C6 to C13 and certain long-chain unsaturated FAs. The activity rate of this enzyme on hexanoic acid, octanoic acid, and decanoic acid were found to be 175.0, 155.0, and 497.0 nmol/min per mg of protein respectively when the enzyme was supplied with 5000 μM substrate. From C13 to C16 there is a downward trend in activity rate until the rate drops to zero for hexadecanoic acid. The enzyme also accepts C18:1 and C18:3 as substrates and can activate the two unsaturated FAs at a rate of 111.8 and 41.2 nmol/min per mg of protein respectively when 30 μM of the FA was supplemented [120]. An unusual feature of this enzyme its substrate specificity range which can be explained by two bell-shaped curves. From C6 to C12 high relative activity rates are 38 observed. From C13 activity rate starts to decrease until reaching zero for hexadecanoic acid. However the activity rate rises again for C18 and C20. RevS from Streptomyces sp. also possesses medium-chain FA specificity. The enzyme has been evaluated has demonstrated activity on C4 to C11 saturated and unsaturated FAs. The enzyme, however, does not accept C13 and longer FFAs as a substrate [121]. The authors furthermore calculated the turnover frequency of RevS which ranged from 5.2 per min for hexanoic acid to 136 per min for octanoic acid. The turnover rate of the enzyme on saturated as well as 2E and 3E monounsaturated FAs was also measured, Figure 3.2. Understanding how internal modifications on the lipid chain will affect an ACS reaction rate can help making an informed decision regarding the ACS options for particular substrate types which would directly link to the CCs being synthesized. 5.218.343.5 44.319.65.515.135.849.519.314.218.241.359.623.60102030405060C6 C7 C8 C9 C10k cat(min -1)Activity Rate of RevS on Saturated and Monunsaturated Medium-chain Fatty AcidsSaturated (E)-2 (E)-3Figure 3.2 Activity Rate of RevS on Saturated and Monounsaturated Medium-chain Fatty Acids RevS has a slight preference towards monounsaturated FAs with a 3E double bond configuration in comparison with 2E. The difference is most noticeable for C6 and C9 FAs. Bar chart drawn using data from Miyazawa et al. (2015). 39 The ability of the aforementioned enzymes to activate branched-chain FAs has not been established. M. tuberculosis FadD6 is one of the few ACSs with proven activity on BCFAs [122]. This enzyme shows remarkably relaxed substrate specificity, with substrates ranging from C3 to C18 FFA, with methyl or hydroxyl modifications on the acyl chain [122]. While these traits seem to make FadD6 an appealing candidate for a CC pathway enzyme, its activity on short and medium BCFAs is unknown. Several ACSs have been evaluated in an E. coli host system. However, the variation in methodologies employed to measure enzyme kinetics, as well as the unavailability of certain enzyme kinetic information, preclude direct comparison between ACS data published in different articles. Side-by-side comparison on a number of ACSs was done Youngquist et al. (2013) including E. coli FadD, M. tuberculosis FadD6, and P. putida PP_0763 for the production of medium-chain fatty alcohols. ΔfadR ΔfadD E. coli harboring FadD expression plasmid grown in LB supplied with dodecanoic acid as the sole carbon source was able to consume 90% of the fatty acid within 8 hours, whereas for the other two ACSs it took at least 11 hours [123]. Hernández Lozada et al. (2020) performed a similar experiment for 1-octanol production in E. coli, comparing the activity of the same set of enzymes for activating octanoic acid supplied to E. coli as the sole carbon source. M. tuberculosis FadD6 consumed more than 80% of the fatty acid 5 hours post-induction while after 12 hours E. coli FadD had consumed about 20% of the fatty acid during the same period [123]. 3.1.3 E. coli-based screen for Acyl-CoA Synthase Specificity A growth-based screening system has been developed to measure ACS activity on different FAs [113, 114, 123]. This system relies on creating an E. coli ΔFadD ΔFadR strain overexpressing an ACS on a plasmid, and growing the strain in minimal media supplied with different FAs as a sole carbon source. FadD, an endogenous E. coli ACS, is removed for plasmid-based ACS control, and FadR is knocked out to allow constitutive transcription of aerobic β-oxidation pathway genes. These include FadL, a long-chain fatty acid transport protein, FadD, a long-chain acyl-CoA synthase, and FadE, an acyl-CoA 40 dehydrogenase. While FadL transports C12 and longer FAs across the membrane, FadE catalyzes the first step of the β-oxidation cycle after the FAs are ligated to CoA by FadD. In the presence of long-chain acyl-CoAs, C12 and longer, as well as excessive amounts of medium-chain acyl-CoAs or FFA, FadR dissociates from its operator sites due to binding of the ligands and changes in the conformation of the protein upregulating fatty acid degradation [124, 125]. It should be noted that acyl-CoA binding abilities of FadR can vary greatly from strain to strain and from species to species [126]. When evaluating ACSs expressed in E. coli, post-translational modification by proteases is another factor that may affect activity. It is noteworthy that E. coli FadD is a substrate for the outer membrane protease, OmpT [127]. Cleaved FadD displays 2-fold higher Km and Vmax compared with intact FadD when using oleate as a substrate, however the catalytic efficacy of both enzymes remain similar [127]. Thus, only the uncleaved enzyme is present in vivo, but in vitro proteolysis of ACSs should be taken into consideration. Certain expression strains such as BL21 (DE3) are OmpT deficient, however based on the available information no issues should arise from expression of intact FadD. 3.1.4 Acyl-CoA Synthase Candidates Relying on the pool of previously-characterized ACSs, we developed criteria for selecting ACSs to evaluate. Our criteria for the acyl-CoA choices was similar to the criteria used for thioesterase, i.e. ability to activate medium to long-chain FAs, confirmed expression in E. coli in functional form, availability of information regarding activity rate. In contrast to thioesterases, the number of options we had for ACS was more limited. We had initially planned on including FadK as a candidate for activating medium-chain FAs, however upon comparison with FadD6, it became evident that FadK has a much lower activity rate. FadD and FadD V451A were included for long-chain and medium-chain FA activation respectively. M. tuberculosis FadD6 had demonstrated activity on BCFAs. Two other ACSs, M. avium Mig and Streptomyces sp. RevS would have also been suitable for CC synthesis, however as we learned about those enzyme later on through our research we did not include them in our constructs. We also selected 41 the C. annum ACS construct as it had been shown to activate 8-methylnonanoic acid in a patent application [72]. Below is a summary of our ACS candidates and their relevant features, designed for expression in E. coli BL21 DE3 ∆FadD ∆FadR, with the goal of determining productivity and substrate specificity in vivo.  C. annum ACS (Unknown FA profile, proven to activate BCFAs according to a patent application)  E. coli FadD (C10 to C18 FA profile, no information regarding BCFA activation)  E. coli FadD V451A (C8 to C18 FA profile, no information regarding BCFA activation)  M. tuberculosis FadD6 (C8 to C18 FA activation, also shown to activate long-chain BCFA) 3.2 Materials and Methods 3.2.1 Designing and Cloning Constructs Double-stranded DNA encoding C. annum ACS was codon optimized and then synthesized by BioBasic Canada. We used DenovoDNA RBS calculator as described in Section 2.2.2. C. annum ACS, E. coli FadD and M. tuberculosis FadD6 were amplified from genomic DNA using their respective ACS primers shown in Supplementary Table 1. M. tuberculosis genomic DNA was generously provided to us by Dr. Yossef Av-Gay lab. The nucleotide sequence of all the expressed genes can be found in Supplementary Table 5. FadD V451A site-directed mutagenesis was performed by PCR. pET28D plasmid was linearized at BamHI and PstI restriction sites. GA was performed afterwards to create the four ACS111, ASC211, ACS212, and ACS311 constructs, Figure 3.3. After sequence verification, plasmids were transformed into NEB T7 Express lysY for protein expression. Colony PCR and sequencing primers have been listed in Supplementary Table 2. Supplementary Table 4 includes a list of the plasmids created in this work and their respective targeted outcomes. The sequence of the expressed genes and the GenBank accession numbers are listed in Supplementary Table 5. Initial attempts to create the knockout strain using lambda phage recombinase were unsuccessful, so we proceeded to using the CRISPR-Cas9 system. Unfortunately, due to time constraints, we were unable to test the ACS constructs. 42 Figure 3.3 Visual Depiction of the Four Acyl-CoA Synthase Constructs 3.2.2 Protein Expression and SDS-PAGE NEB T7 Express lysY cells harboring the ACS211, ACS212, and ACS312 constructs were plated on LB agar supplied with 50 μg/mL Kan. Empty pET28D vector was used as a negative control. Single colonies were picked from the plate and used to inoculate 3mL fresh LB in culture tubes. Cultures were grown at 30 °C. At OD 0.5-0.9 cells were induced with 0.5 mM IPTG and allowed to grow at 30 °C in a shaker set at 280 rpm. ACS111 construct overexpressing C. annum acyl-CoA synthase was chilled to 15 °C prior to induction and then grown at 15 °C for 24 hours after induction. For lysis the 3mL culture was transferred into 1.5mL centrifuge in two separate batches and centrifuged. Cells were pelleted at 21,000g for one minute, supernatant was subsequently discarded. The pellet was resuspended and washed with PBS buffer. Afterwards cells were resuspended in 300 μL PBS (pH 7.2) buffer. Tubes were placed on ice and cell disrupted was performed by ultrasonication for 5 min (cycles with 10 sec work and 10 sec pause) at 30% amplitude. After centrifugation at 21,000g soluble fraction of the cell lysate was transferred into a separate tube, and protein concentrations were measured using NanoDrop 2000. Protein denaturation for gel electrophoresis was performed using Bolt™ Bis-Tris Plus Mini Gels according the manufacturer’s protocol. Approximately 20 μg of protein mixture was transferred into 0.2 mL PCR tubes, 4X Bolt™ LDS Sample Buffer and dH2O were then added to make the total sample volume 10 μL. Afterwards the samples were incubated at 70 °C for 10 minutes. MOPS SDS running buffer was used for electrophoresis. PageRuler™ Prestained Protein Ladder was used for size determination. Following the 43 FadD and FadD V451A 62.3 KDa Empty Vector ACS FadD V451A FadD FadD6 100 KDa 70 KDa 55 KDa FadD6 64.3 KDa 40 KDa staining procedure, the gels were scanned and the produced image was modified using Paint.NET to increase contrast and enhance the visual effect of band intensities. 3.3 Results and Discussion Figure 3.4 shows the outcome of our SDS-PAGE analysis. We could not detect any bands corresponding to C. annum ACS which is 73.6 KDa. The two bands appearing in the FadD expression vectors are distinct from the empty vector and the other two ACS constructs. E. coli FadD is 62.3 KDa, however the bands are located at ~ 70 KDa, slightly higher than expected. Our initial plan was to evaluate ACS constructs using an E. coli growth-based screening method, meaning that the cells are supplied with a particular FA as a sole carbon source and then growth rates are monitored. E. coli with ∆FadD and ∆FadR knockouts would only be able to grow if they were able to metabolize the FA provided. While three of the four ACS constructs yielded bands near their appropriate molecular masses, for the E. coli FadD constructs, the band location was ~ 8 KDa too high. It is possible Figure 3.4 SDS-PAGE Results from Acyl-CoA Synthase Constructs No bands could be detected for C. annum ACS, E. coli FadD V451A and FadD had bands with a higher molecular weight than expected. A band corresponding to M. tuberculosis FadD6 is present in the gel, however due to the presence of a similar in the empty vector further experiments need to be conducted to confirm results. 44 that C. annum ACS was insoluble in PBS buffer and we would have been able to obtain a band by testing the insoluble fraction. One possibility is that incomplete denaturation had resulted in higher molecular weight bands: Ford et al. (2015) had also conducted SDS-PAGE analysis of FadD and obtained the correct mass, and they used a reducing agent during the denaturation step. The Bolt™ LDS sample buffer used in our lysis protocol did not contain a reducing agent and the compound must be added separately. FadD6 appears to have a band corresponding to the appropriate molecular weight. A similar-sized band also appeared in our negative control although with lesser intensity, however this band does not exist in the rest of the ACS constructs. Given these results, it is difficult to conclude with high certainty that our FadD6 strain is successfully expressing the protein. 3.4 Future work To address the higher than expected molecular mass of FadD and FadD V451A, first recommendation is to use reducing agents such as 2-Mercaptoethanol in the denaturing stage. This protein has successfully been expressed and analyzed via SDS-PAGE in at least two publications [113, 118]. With regards to the FadD6 construct, given the absence of a similar sized band in other ACS constructs as well as the intensity of the FadD6 band being higher than the similar sized band in the negative control, it is likely that the band in our SDS-PAGE analysis corresponds to the FadD6 protein. However, to be certain, the protein can be His-tagged and then purified using affinity chromatography to yield more accurate results. After confirming soluble expression of the above proteins, they need to be transformed into an E. coli strain with ∆FadD and ∆FadR knockouts. Subsequently, the growth rate of E. coli overexpressing ACS constructs on BCFAs can be evaluated. This could reveal the extent to which the location of the methyl branch affects the catalytic activity of the ACSs being tested. To our knowledge these experiments have not been performed previously, and it would be a novel addition to prior findings 45 about these ACSs. Amongst the studied bacterial ACSs, there are very few with proven activity BCFAs. If FadD is unable to activate BCFAs, an alternative strategy would be to search for ACSs within bacterial species that are known BCFA producers using the sequence of the FACS and M-domain motifs discussed in the introduction section. For example, LcfA and LcfB are predicted to encode long-chain fatty acyl-CoA synthases in B. subtilis, and similarly to FadD the two enzymes are involved in the β-oxidation pathway [128]. Mig and RevS ACSs mentioned in this chapter also belong to strains capable of producing BCFAs. M. bovis and Streptomyces sp. have both shown the synthesize BCFAs [129, 130]. For CC synthesis, the choice of ACS will be one of the determinants of the acyl-chain length of the final product. Based on prior results E. coli FadD can act as a gate keeper for the synthesis of CC with C10 and longer acyl-chain. FadD has very low activity on octanoic and shorter-chain fatty acids. On the other hand FadD6 has a broader substrate specificity making it suitable for CCs with varying acyl-chain lengths. Mig and RevS ACSs can also be suitable for CC synthesis as they possess specificity towards medium-chain FAs. Overall, development of TE and ACS panels, carrying variants with differing substrate specificities, could enhance control and modularity of the capsaicinoid and capsinoid pathways. 3.5 Conclusion In this chapter we attempted to express a number of ACSs which could potentially be used for CC synthesis. The thioesterase and acyl-CoA synthase together determine the composition of the acyl-CoA pool available to capsaicin synthase. Although we could not appropriately test the ACSs in this section, the panel of ACSs evaluated for this system are well-positioned to demonstrate proof of concept for this principle. Furthermore, in designing this experiment we learned that there is room for research regarding substrate preference of ACSs as they have not been directly compared and tested against different BCFAs. 46 Chapter 4 Production of Vanillylamine and Vanillyl Alcohol 4.1 Introduction Bioconversion of vanillin to vanillylamine in E. coli requires heterologous expression of a transaminase with an amino donor and pyridoxal phosphate as cofactor. On the other hand, catalysis of vanillin to vanillyl alcohol can be done using the endogenous reductase and dehydrogenase host enzymes. Both substrates can then be used for CC biosynthesis. Transaminases have gathered considerable attention due to their potential applications in pharmaceutical, agrochemical, and fine chemical industry [131]. Valorization of lignin and use of renewable feedstock is another potential application of transaminases [69]. To achieve bioconversion of vanillin to vanillylamine in E. coli, we selected CV2025 ω-transaminase from C. violaceum on the basis of previous experimental evidence demonstrating transaminase activity of the enzymes in E. coli as well in P. putida [69, 132]. In this chapter we attempt soluble expression of C. violaceum CV2025 and C. chinense pAMT transaminase, as well discuss avenues for vanillyl alcohol production in E. coli. 4.1.1 Production of Vanillylamine and Enzyme Candidates CV2025 transaminase has already been shown to convert vanillin to vanillylamine in E. coli [69]. Du et al. (2014) showed that amongst eight lignin breakdown intermediates, vanillin had the fastest initial reaction rate. Furthermore the effects of different MBA amino donor and vanillin concentrations were studied to establish kinetic models for the enzyme. Cell lysate experiments demonstrated that at vanillin concentration below 10 mM, an MBA to vanillin ratio > 2:1 had the optimum results in which close to 100% bioconversion could be achieved within two hours. In whole-cell bioconversion experiments, 89% vanillylamine yield was achieved within 24 hours when the cells were supplied with 20mM MBA, 0.2mM PLP cofactor, and 5mM vanillin. At higher than 10 mM vanillin concentrations, the authors recommended that a 4:1 MBA to vanillin ratio could result in high conversion rates. However, increasing the vanillin concentration would eventually lead to product inhibition and increased back 47 reaction rate. To keep the reaction equilibrium towards vanillylamine production, the ratio of vanillin to amino donor need to be taken into consideration. CV2025 has also been expressed in P. putida and compared against a panel of other transaminases for vanillin to vanillylamine bioconversion [132]. In this paper the authors initially developed a growth-based screening platform was developed to evaluate the ability of P. putida KT2440, expressing different transaminase, for growth in M9 medium supplemented with vanillylamine as the sole carbon source. Two enzymes with the highest growth rate were identified P. putida SpuC-II and C. violaceum CV2025. Afterwards the activity rate of the two transaminases were compared in whole-cell bioconversion experiment. Pp-SpuC-II and CV2025 produced 0.70 ± 0.20 mM (in 7 hours) and 0.92 ± 0.30 mM (in 2 hours) vanillylamine respectively when using 10 mM vanillin as the substrate and 100 mM of alanine as the amine donor. Vanillylamine concentration begins to drop after reaching maximum levels due to product assimilation. In the bioconversion experiment, vanillic acid and vanillyl alcohol were detected as unwanted side-products [132]. Overall, the superior performance of CV2025 in multiple microbial systems as well the availability of kinetic data for this enzyme makes it a salient choice for our system. We decided to create a construct for pAMT as well to determine whether we can express this plant based transaminase in soluble form. The catalytic activity of pAMT, responsible for the transaminase reaction in Capsicum species has been confirmed through in vitro experiments [70]. Previous reports have claimed that the transamination of vanillin to vanillylamine by pAMT was impossible to detect in E. coli BL21 (DE3) cell-free lysates [70]. The calculated Gibbs free energy of the reaction had a positive value and it was theorized that conversion of vanillin to vanillylamine was thermodynamically unfavorable in vitro. Hence a product removal system was needed for vanillin to vanillylamine conversion to proceed. Consequently, the enzyme was evaluated in carrying the reverse reaction using pyruvate as an amine acceptor, and results showed successful transamination of 48 vanillylamine to vanillin. It is noteworthy to mention that no enzyme activity was detected when induction was done at 37°C, however reducing the temperature to 15°C resulted in the expression of the enzyme in active form. 4.1.2 Production of Vanillyl alcohol Aromatic aldehydes are rapidly reduced to their corresponding alcohol by the endogenous enzymes in E. coli [133]. Vanillin is toxic to microorganisms and to alleviate its deleterious effects both E. coli and S. cerevisiae convert it to less toxic compounds such as vanillyl alcohol [133, 134]. Many of the research papers on vanillin production and utilization in E. coli also report synthesis of vanillyl alcohol as an unwanted by-product [71, 132, 135]. Vanillin detoxification processes can be achieved through either reduction or oxidation of vanillin, and appears to be carried out by a variety of genes. Kunjapur et al. (2014) reported that the deletion of three aldo-keto reductase genes, and three alcohol dehydrogenase genes from E. coli MG1655(DE3) substantially decreased the conversion of vanillin to vanillyl alcohol. In 24 hours, in contrast to the full conversion achieved by the WT strain, there were no detectable levels of vanillyl alcohol in the knockout strain [133]. These results suggest that a vanillylamine-producing E. coli strain, for capsaicinoid production, would require these three aldo-keto genes to be knocked out, while a vanillyl-alcohol-producing E. coli strain, for capsinoid production, already possesses the requisite genes. Decreasing media pH below 7.0 has also been reported to improve the vanillin to vanillyl alcohol conversion, as the substrate is protonated at a faster rate [135]. However, alternative approaches, like overexpressing the three aldo-keto genes, may be preferable given the suboptimality of low pH culture conditions for the rest of the CC pathway. 4.2 Materials and Methods 4.2.1 Designing and Cloning Constructs Double-stranded DNA encoding CV2025 and pAMT transaminase were codon-optimized and ordered through Genewiz. We used DenovoDNA RBS calculator as described in Section 2.2.2. pET28D 49 plasmid was linearized using BamHI and PstI. The two transaminases were amplified using their respective VA111 and VA211 primers shown in Supplementary Table 1. Afterwards GA was performed to create the VA111 and VA211 constructs, Figure 4.1. After transformation into Trans1-T1 competent cells, sequence verification was performed. Supplementary Table 2 shows the list of primers used for sequencing and colony PCR. NEB T7 Express lysY were then transformed with the plasmids for protein expression analysis. Detailed features of the constructs and strains used in this work can be found in Supplementary Table 4. The sequence of the expressed genes and the GenBank accession numbers are listed in Supplementary Table 5. Figure 4.1 Visual depiction of the Transaminase Constructs 4.2.2 Protein Expression and SDS-PAGE Samples were prepared for SDS-PAGE as described in section 3.2.2, with the following changes: the VA211 construct overexpressing pAMT transaminase was chilled to 15 °C prior to induction, and then grown at 15 °C for 24 hours after induction. VA111 overexpressing CV2025 transaminase was grown and induced at 30 °C. 4.3 Results and Discussion SDS-PAGE analysis of the two transaminase constructs show soluble expression of the CV2025 transaminase (51.2 KDa), Figure 4.2. A strong bands corresponding to the molecular weight of the transaminase is visible in the gel. We could not detect any bands corresponding to pAMT (50.7 KDa). 50 CV2025 51.2 KDa Empty Vector pAMT CV2025 55 KDa 70 KDa 40 KDa Figure 4.2 SDS-PAGE results of pAMT and CV2025 Transaminase A strong band corresponding to the molecular weight of CV2025 was detected in the soluble fraction of the lysate, however pAMT overexpressing strain did not produce any distinctive bands. Given that our pAMT and CV2025 constructs both share the same RBS, and translation rates were predicted using DenovoDNA, we believe it is unlikely for the translation of the protein to be an issue. Our next steps would have been to analyze the insoluble fraction using SDS-PAGE, as well as His-tagging the pAMT to permit its purification and concentration. Due to time constraints, we were unable to test the CV2025 transaminase for its activity in E. coli. However, soluble expression suggests that the transaminase is in its active form. 4.4 Future Work The next step in construct testing would be to verify that CV2025 can enable conversion of vanillin to vanillylamine. This would confirm that the encoded enzyme is functional. To test vanillylamine conversion, amino donors such as MBA, GABA or L-alanine could be supplied in the growth media along with vanillin and PLP cofactor [69]. The conversion of these precursors to vanillylamine can be determined through HPLC or GC-MS analysis [69, 136]. Given the high yield of the CV2025 transaminase, 51 which Du et al. (2014) quantified to be 689 mg/L (4.45 mM) vanillylamine in 24 hours from 761mg/L (5mM) vanillin, it is unlikely for vanillylamine production to be a bottleneck in capsaicinoid biosynthesis pathway. Vanillyl alcohol production can be achieved by simply feeding vanillin to WT E. coli and allowing the cells to grow for 24 to 48 hours in plain LB. Vanillin is toxic to E. coli and the compound is naturally reduced to vanillyl alcohol by the endogenous aldo-keto reductases including DkgB, YeaE, and DkgA [133]. High-acidity conditions cause faster protonation of vanillin, leading to faster production of vanillyl alcohol, so growth media pH screens could be used to determine whether a lower pH could benefit the in vivo vanillin conversion rate without interfering with the rest of the capsinoid pathway enzymes. In past work, knockouts removing the reductases from the genome and overexpressing them from a plasmid enabled best control over conversion. The same knockout strain can be used for capsaicinoid production, but instead of a reductase, CV2025 will be overexpressed on a plasmid. Kunjapur et al. (2014) demonstrated that the endogenous E. coli reductases were able to convert ~80% of vanillin to vanillyl alcohol within 48 hours when supplied with ~5mM vanillin. Hence vanillyl alcohol production is also unlikely to a bottleneck in capsinoid production pathway. 4.5 Conclusion In this chapter we discussed potential pathways for vanillylamine and vanillyl alcohol production. Furthermore, we demonstrated soluble expression of the CV2025 transaminase. Although we did not evaluate vanillylamine production, previous publications have provided strong evidence that CV2025 is a suitable transaminase for vanillin. Endogenous enzymes in E. coli can catalyze the reduction of vanillin to vanillyl alcohol, however the same endogenous enzymes could divert vanillylamine production towards vanillyl alcohol. Therefore, creating a knockout strains would be good steps for enhancing capsaicinoid production. 52 Chapter 5 Capsaicin Synthase and the Condensation Reaction 5.1 Introduction Capsaicin synthase catalyzes the formation of CCs by condensing an acyl-CoA with vanillylamine or vanillyl alcohol, and is dependent on ATP and Mg2+ [137]. This enzymes belongs to the BAHD acyltransferases superfamily, primarily present in bacteria, algae, and plants [141]. Only Capsicum species harbor and express capsaicin synthase. Capsaicinoids play an important role in the food and pharmaceutical industries and a number of researchers have been engaged in improving their production by optimizing pepper plant cultivation conditions, chemical synthesis, enzymatic synthesis or methods such as tissue culture [137]. Biosynthetic production of capsaicinoids would require functional expression of capsaicin synthase in addition to the rest of the pathway enzymes. The only available information regarding heterologous expression of capsaicin synthase in E. coli is in a patent, although a student’s thesis also mentioned functional expression of this enzyme in S. cerevisiae [72, 138]. In this chapter, we describe creation of four constructs, two are to test for capsaicin synthase solubility with and without the SUMO tag, and the other two are for capsaicinoid production with >=C10 and >=C6 acyl chain lengths. 5.1.1 BAHD Enzyme Family BAHD members are involved in the biosynthesis of a number of important drugs, such as cocaine, morphine, paclitaxel, and vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine [139, 140]. They are CoA dependent and are responsible for producing volatile esters, anthocyanins, lignin, phenolics, alkaloids, as well as defense compounds and phytoalexins [141]. In the plant kingdom phylogenetic trees, capsaicin synthase has been classified in Clade III with other acyltransferases involved in the synthesis of volatiles in flowers and ripening fruits, such as SALAT from P. somniferum catalyzing the synthesis of morphinan-type benzylisoquinoline alkaloids, DAT from C. roseus which produces vandoline, and EcBAHD7 from E. coca catalyzing cocaine [140, 141]. 53 BAHDs have relaxed substrate selectivity and can accept various acyl-CoAs, however, they are more specific towards the acyl acceptors [142]. Electrostatic properties and hydrogen-bonding interactions have shown to influence the substrate specificity of the acyl acceptor binding pocket [143, 144]. Moreover, it has been noted that most acyltransferases within Clade III accept a wide range of alcohol substrates [141]. Capsaicin synthase belongs to this clade, and it could possibly accept alcohol substrates beyond just vanillyl alcohol. Coniferyl esters, and specifically capsiconiate and dihydrocapsiconiate have been isolated from C. baccatum [145]. There is a possibility that capsaicin synthase could carry out the condensation reaction between coniferyl alcohol and an acyl-CoA. If the substrate specificity is sufficiently relaxed, it might be possible to produce additional compound libraries by simply changing the aromatic moiety. For BAHD acyltransferases identified through genetic profiling, substrate specificity predictions based on sequence homology have had limited success [141]. Therefore, biochemical assays have been recommended as the preferred tool to assess the activity of BAHD members. A number of BAHD acyltransferases have been heterologously expressed in E. coli and characterized. For example, acetyl-CoA:benzylalcohol acetyltransferase (BEAT) from C. breweri was expressed in E. coli in active form, successfully synthesizing benzylacetate [146]. The authors evaluated a range of acyl donor and acyl acceptor substrates to assess the substrate specificity of BEAT. In another study, piperamide synthase from black pepper was functionally expressed in E. coli and a wide array of CoA-donors and amine acceptors were evaluated [147]. Overall, expression of BAHD members in E. coli has been successfully attempted a number of times, but none utilizing vanillylamine have been demonstrated. 54 BAHD proteins typically have 420-460 amino acids [146]. A number of conserved domains are present in the family. The two most important are the HXXXDG motif common in acyltransferases that accept CoA thioesters as substrates, and the DFGWG motif located near the C-terminus [139]. Of the 717 BAHD genes identified in Rosaceae species, all of them contained only one of the motifs [148]. Crystal structure of vinorine synthase revealed that the DFGWG motif is actually located away from the catalytic site and appears to solely play a role in the conformation of the enzymes [139]. Figure 5.1 illustrates the alignment of the two conserved motifs across the BAHD enzymes mentioned in this chapter. Capsaicin synthase (AT3) has not been fully characterized and evaluated, however all the evidence gathered so far supports the hypothesis that AT3 is responsible for CC synthesis. AT3 is present in C. annum bell peppers, however a 2.5kb deletion in the gene which spans the putative promoter region is linked to the absence of protein transcripts [58]. This deletion correlates with the absence of pungency in the plant fruits. Furthermore, the 2.5kb deletion does not exist in any of the pungent variants sequenced [58]. In a subsequent study, a truncated AT3 allele was identified and linked to a four base deletion resulted in a frameshift mutation [149]. The truncated AT3 has also been linked to a loss of pungency [149]. Evolutionary studies performed on AT3 within the Capsicum genus and Solanaceae family revealed that the coding region of AT3 is highly conserved [150]. Despite it being a plant enzyme, we a) b) Figure 5.1 The Two Main Motifs of the BAHD Family Capsaicin synthase from C. annum, cocaine synthase from E. coca, vinorine synthase from R. serpentine, Acetyl-CoA-benzylalcohol acetyltransferase (BEAT) from C. breweri, and piperamide synthase from P. nigrum. a) HXXXDG motif highlighted in red b) DFGWG motif highlighted in red. About 66% of the identified BAHD members carry at least one of the motifs. 55 chose to use this BAHD for the heterologous CC pathway, since it the most likely of the BAHDs published to use vanillylamine as a substrate. 5.1.2 Capsaicin Synthase Solubility Information from publications had stated that recombinant expression of AT3 had been met with challenges due to its insolubility [58]. We conducted further research to identify potential solutions in advance. Fusion systems have been reported as the most successful at improving solubility of recombinant proteins [151]. Fusion tags can typically help with improving solubility, expression, and purification [151]. A number of fusion genes have been developed including GST, NusA, MBP, and others [151]. We selected the SUMO tag in particular due extensive number of publications utilizing this gene demonstrating higher performance in improving solubility [152, 153]. Furthermore, usage of the SUMO tag has been mentioned in a patent application for expression of capsaicin synthase in E. coli [72]. In the patent, SUMO-AT3 was inserted in a pET expression vector. Induction was done with 0.5 mM IPTG, and growth temperature and during were set at 16 °C and 20 hours respectively. We attempted to replicate these conditions to find out if whether we can achieve similar results. 5.2 Materials and Methods 5.2.1 Designing and Cloning Constructs C. chinense capsaicin synthase AT3 was codon optimized and then ordered through BioBasic Canada. The pCIOX plasmid was ordered from Addgene for SUMO tag amplification. We used DenovoDNA RBS calculator as described in Section 2.2.2. CS1111 construct was created by inserting AT3 under T7 promoter in pET28D, Figure 5.2 a). Initially SF211 plasmid was linearized using XhoI/PacI and AT3 amplified using CS1111 AT3_F+pET28D/CS1111 AT3_R+pET28D primers, the sequence of which can be found in Supplementary Table 1. Using GA the two fragments were joined. CS1112 construct was made by the addition of the SUMO tag to AT3, Figure 5.2 b). SUMO tag was amplified using CS1112 SUMO_F+pET28D/CS1112 SUMO_R+AT3_R and AT3 amplified using CS1112 AT3_F+SUMO_R/ CS1112 56 AT3_R+pET28D. The two fragments were ligated with the SF211 linearized vector using GA. CS2112 construct was created by placing AcTesA and FadD under a single T7 promoter and SUMO-AT3 and CV2025 under a second T7 promoter. This was done to reduce the overall length of mRNA being generated. FadR was also placed under LacI promoter. CS2212 was created from CS2112 by switching FadD with FadD6, Figure 5.2 c). We placed restriction sites on the 3’ and 5’ ends of all the genes to facilitate switching of enzymes for future experiments. Assembled constructs were initially transformed into Trans1-T1 competent cells. After overnight growth successful clones were identified using colony PCR. They were then inoculated for plasmid extraction. Supplementary Table 2 includes the sequence of primers used for sequencing and colony PCR. After sequence verification, the plasmids were transformed into T7 Express lysY for protein expression analysis. Supplementary Table 4 includes a list of the plasmids created in this work and their respective targeted outcomes. The sequence of the expressed genes and the GenBank accession numbers are listed in Supplementary Table 5. Figure 5.2 Visual Depiction of Capsaicin Synthase Constructs a) and b) Capsaicin synthase expressed without and with the SUMO tag. C) Capsaicinoid production constructs with the goal of synthesizing >=C10 Capsaicinoids using FadD and >=C6 Capsaicinoids using FadD6. a) b) c) 57 5.2.2 Protein Expression and SDS-PAGE Samples were prepared for SDS-PAGE as described in section 3.2.2. However, capsaicin synthase constructs CS1111 (AT3) and CS1112 (SUMO-AT3) were chilled to 15 °C prior to induction, and then were grown at 15 °C for 24 hours after induction. Protein denaturation was performed with and without 8M urea prior to incubation at 70 °C. 5.3 Results and Discussion It has previously been reported that capsaicin synthase is extremely insoluble in E. coli [58]. However, we identified information in a patent suggesting that the SUMO tag made it possible to express capsaicin synthase in soluble form [72]. We followed the same expression conditions, for both SUMO-tagged and untagged forms. No distinctive bands were visible in the soluble fraction in the SDS-PAGE for SUMO-AT3 (60.5 KDa) and AT3 (49.3 KDa), Figure 5.3. Figure 5.3 AT3 and SUMO-AT3 SDS-PAGE Results The denaturation step was performed with and without 8M urea. In all the tested conditions no bands corresponding to the molecular weight of AT3 nor SUMO-AT3 could be detected. 55 KDa 40 KDa 70 KDa Empty Vector AT3 AT3 (with 8M urea) SUMO-AT3 SUMO-AT3 (with 8M urea) 58 We attempted protein denaturation in reducing conditions with 8M urea, however the results remained inconclusive. Similar to previously designed constructs, the DenovoDNA RBS Calculator predicted reasonable translation rates for AT3 [106]. Therefore, further investigation is necessary to identify whether mRNA is being transcribed using methods such as qPCR, or whether small amounts of soluble protein are being expressed in functional form. Even small amounts which would go undetected in the protein gel could potentially result is successful catalysis of capsaicinoids. 5.4 Future Work Subsequent to ensuring that mRNA is being transcribed, experiments can be performed to troubleshoot the expression and function of this protein. If the protein is being translated in low rates making it hard to detect on SDS-PAGE, His-tagging could be used to purify and concentrate AT3 prior to analysis. It is also possible that the sample preparation protocol does not denature or solubilize the protein, hence testing the activity of the enzyme might a good next step. A cell-free enzyme assay can be performed by expressing the AT3 and SUMO-AT3 constructs, and adding an acyl-CoA preferably C9 or C10 and vanillylamine to the lysate. Subsequently, analysis can be performed by HPLC or GC MS. Chaperone co-expression may also enable AT3 expression. Other BAHD enzymes, namely the shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1), were only soluble when GroEL and GroES chaperon proteins were co-expressed [154]. Finally, an alternative host such as P. pastoris may be used, if all other attempts at expression fail. This was also the case for another related acyltransferase. Expression of the acyltransferase responsible for acyl sucrose acetylation in tomato trichomes, was initially attempted in E. coli [155]. However, only expression in P. pastoris enabled soluble protein expression. Once established, expression of the capsaicin synthase constructs could enable evaluation of capsaicin synthase substrate specificity, which is unlikely to be stringent, given the specificities of other enzymes from the same BAHD family. Provided substrate tolerance is sufficiently moderate, synthetic precursors could be used 59 with capsaicin synthase to demonstrate the modularity principle envisioned in this thesis. 5.5 Conclusion In this section, we sought to evaluate the expression of the C. chinense capsaicin synthase, AT3. A number of plant-based BAHD enzymes have been expressed in E. coli and the ones that encountered problems with solubility either used the SUMO tag, chaperons or simply used another host. Although we attempted AT3 expression for both native and SUMO-tagged forms, no expression was visible by SDS-PAGE for either of the constructs. These results are preliminary and a number of steps can be taken to address the inconclusive results. Unlike other enzymes in the CC synthesis pathway, capsaicin synthase does not bacterial alternatives, but its expression is crucial in having a functional pathway. 60 Chapter 6 Summary and Conclusion This thesis sought to evaluate E. coli as a chassis for the synthesis of CCs, which have bioactive properties and other well-established uses. A microbial platform for CC production could enable a more rational investigation of natural and new-to-nature CCs, and potentially enable scaled-up production. Heterologous CC pathway construction in E. coli requires introduction of a minimum of 3 genes for capsinoid synthesis: A thioesterase, an acyl-CoA synthase, and finally a capsaicin synthase. Addition of a transaminase is needed for capsaicinoid synthesis, and branched-chain CCs require two additional genes. Other modifications might be needed to direct flux towards CC synthesis, e.g. overexpression of FadR transcription regulator and deletion of the β-oxidation pathway genes, FadD and FadE. In chapter two, constructs with four different thioesterases were prepared. Coexpression of BKD with AcTesA’, a novel combination in E. coli, yielded medium BCFA, but surprisingly also yielded large quantities of short BCFA. While the cause of this is unclear, the two proposed mechanisms include an ACP binding pocket which excludes BCFA, as well, better positioning the thiol group near the TE active site due to the presence of methyl branches. Chapters three to five focused on the selection and preparation of constructs for the other three pathway enzymes. Based on prior publications, FadD6 appears to be the most suitable for BCFA activation, however its activity rate on medium BCFA is unknown and other ACSs may need to be tested. CV2025 has shown transaminase activity in E. coli and we were able to demonstrate expression of this protein in soluble form. Furthermore, vanillyl alcohol production has previously been confirmed by a number of publications. We were unable to detect soluble expression of Capsicum-based proteins, ACS, pAMT, and AT3. Although ACS and pAMT have bacterial alternatives, expression of AT3 requires further investigation as it is a key enzyme in the biosynthesis pathway. We had limited success in replicating previously-published results. However, there is ample evidence that production of fatty acids, activation of the fatty acids, as well as production of 61 vanillylamine and vanillyl alcohol are all feasible in E. coli, and to this point this host can be considered as suitable chassis for CC synthesis. However, the main bottleneck, which is expression of capsaicin synthase in soluble form, is yet to be resolved. A number of experiments have been recommended, including cell-free enzyme assays, as well as co-expression of chaperons. The results can then provide an answer to whether E. coli can be considered as suitable host for CC synthesis. 62 Chapter 7 Future Work 7.1 Eliminating Dependency on non-natural vanillin One of the short comings of this system is its reliance on vanillin as a supplied precursor. Therefore, different precursors and pathways which enable vanillin biosynthesis could be considered as alternative sources to exogenously-supplied vanillin. Numerous biosynthesis pathways for vanillin have demonstrated in E. coli. Use of the shikimate pathway for aromatic amino acid production along with five non-native enzymes enabled vanillin synthesis from L-tyrosine at 97.2 mg/L, and from glucose at 19.3 mg/L [74]. Ferulic acid, which can be extracted from agricultural waste, can also be used as an alternative starting precursor [156, 135]. Luziatelli et al. (2019) developed a recombinant E. coli strain capable of converting ferulic acid to vanillin. The pathway involved heterologous expression of two genes from Pseudomonas: feruloyl-CoA synthetase and feruloyl-CoA hydratase, both of which were integrated into the E. coli genome [135]. The authors also performed optimization on bioconversion conditions, as well created a system for controlled release of ferulic acid. The end result was one of the highest vanillin titers achieved in literature, 4.3 g/L. However, the price of ferulic acid has been reported as a drawback for these bioprocesses [157]. Lignin has also been investigated as a potential feedstock for vanillin production. Being the most abundant carbon-based material in nature, lignocellulosic biomass represents an attractive renewable source for aromatic building blocks. Lignin, a major component of lignocellulosic biomass, is mainly composed of three monolignols: p-coumaryl, coniferyl, and sinapyl alcohol [158]. These are linked together into a recalcitrant matrix through C-C and C-O bonds via a radical coupling reaction. While chemical depolymerisation has been successful in producing high yields of aromatic lignin monomers, biological methods are also gaining interest, and particularly the use of biological pathways to funnel lignin depolymerisation monomers into industrial chemicals [159]. Since vanillin may be derived from lignin, it could also be used as a precursor source for CC. 63 7.2 Capsaicinoid Antimicrobial Properties and Their Inhibitory Effects Capsaicinoids have antimicrobial properties and to address potential toxicity of capsaicinoids on the engineered strain, transporter proteins can be expressed to reduce capsaicinoid concentrations inside the cell. Studies have been performed to determine the impact and inhibitory effects of capsaicin across microorganisms. A detailed study was performed on S. cerevisiae by Kurita et al. (2002). It was shown that at 500 ppm capsaicin increased the lag time of S. cerevisiae cells by nine hours, reduced growth rate by approximately half, and reduced overall cell density by about 15-20% [160]. At 100 ppm, the impact was minimal. It was suggested that PDR5, a multi-drug transporter, is responsible for pumping capsaicin outside of the cell. In the presence of 250 ppm capsaicin in growth media, expression of PDR5 was four times higher than control conditions. In addition, deletion of this gene increased the lag period from 7 hours to 29 hours in the presence of 1600 ppm capsaicin 160. However, capsaicin does not always have a growth inhibition effect: In an interesting finding, addition of 25 to 50 ug/mL of capsaicin to S. marcescens growth media resulted in increased growth compared to control [161]. The authors could not provide an explanation for this observation, there is a possibility that capsaicin was somehow being metabolized. Kar et al. 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Asian Pacific Journal of Tropical Disease, 6(10), 807–810. 72 Appendices Supplementary Table 1. Primers Used in this Study Primer Description Sequence MCS1_F For amplifying pACYCDuet-1 MCS. Used to create pET28D construct. GCGGATAACAATTCCCCTCTAGCATATGGCAGATCTCAATTGGATATCG MCS1_R For amplifying pACYCDuet-1 MCS. Used to create pET28D construct. GGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAACCCGGTCGTCGG MCS2_F For amplifying pACYCDuet-1 MCS. Used to create pET28D construct. GGAATTGTGAGCGGATAACAATTCCCCATGGGCAGCAGCCATCAC MCS2_R For amplifying pACYCDuet-1 MCS. Used to create pET28D construct. CCCAAGGGGTTATGCTAGTTATTGCTCAATTTCGATTATGCGGCCGTGTAC SF111 TesA’_F+p28D For amplifying E. coli TesA’ from genomic DNA. Used to create SF111, SF112, BF111, and BF112 constructs. CCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTAGAGCAGGGAGGTAACAAGATGGCGGACACGTTATTGATTCTGG SF111 TesA’_R+p28D For amplifying E. coli TesA’ from genomic DNA. Used to create SF111 and SF112 constructs. GCTCAGCGGTGGCAGCAGCCTAGGATCTTAATTAATTATGAGTCATGATTTACTAAAGGCTGCAAC SF211 FadR_F+p28D For amplifying E. coli FadR from genomic DNA. Used to create SF211 construct. GGCGCGGGGCATGACTATCGTCGCTAGCAGGGGGTAATATGGTCATTAAGGCGCAAAGCC SF211 FadR_R+p28D For amplifying E. coli FadR from genomic DNA. Used to create SF211 construct. ATGCGCACCCGTGGGGCCGCCATGCCGGCTTATCGCCCCTGAATGGCTAAATCACC ecTesA_F (Y145K) For generating Y145K mutation in TesA’ using PCR site directed mutagenesis. Used to create SF112 and BF112 construct. GAAGAGGTCAAACTCAAGCCACAATGGATGCAGGATGACG ecTesA_R (Y145K) For generating Y145K mutation in TesA’ using PCR site directed mutagenesis. Used to create SF112 and BF112 constructs. GGCTTGAGTTTGACCTCTTCCATAAAAAAGGGCAGCAGCG SF211 AcTesA’_F+p28D For amplifying A. bayly AcTesA from synthesized DNA fragment. Used to create SF211 and BF211 constructs. GCGATCGCTGACGTCGGTACCCTCGAGAGTAAGGAGCTTATTATATGGGC SF211 AcTesA’_R+p28D For amplifying A. bayly AcTesA from synthesized DNA fragment. Used to create SF211 construct. GCTCAGCGGTGGCAGCAGCCTAGGATCTTAATTAATTACAGCGCGCCTTTGATATAC SF311 CvFatB1_F+p28D For amplifying C. viscosissima FatB1 from synthesized DNA fragment. Used to create SF311 and BF311 constructs. TCGCTGACGTCGGTACCCTCGAGCGCATTAAGGAGGTTTTTTTATGGTTGCTGCTGCTGCTACC SF311 CvFatB1_R+p28D For amplifying C. viscosissima FatB1 from synthesized DNA fragment. Used to create SF311 construct. GTGGCAGCAGCCTAGGATCTTAATTAATTAAGAAACAGAGTTACCGTTAGAGGTTTTAGC BF111 TesA’_R+FabH2_F For amplifying E. coli TesA’ from genomic DNA. Used to create BF111 and BF112 constructs. ACCCCCTATGAGTTGTTAATTAATTATGAGTCATGATTTACTAAAGGC BF211 AcTesA’_R+FabH2_F For amplifying A. bayly AcTesA from synthesized DNA fragment. Used to create BF211 construct. ATACCCCCTATGAGTTGTTAATTAATTACAGCGCGCCTTTGATATACG BF211 FabH2_F+Thio_R For amplifying B. subtilis FabH2 from genomic DNA. Used to create BF211 construct. TTAATTAACAACTCATAGGGGGTATTTGTTATGTCAAAAGCAAAAATTACAGCTATCGGC BF211 FabH2_R+BKD_F For amplifying B. subtilis FabH2 from genomic DNA. Used to create BF211 construct. CCTTTCTTCCTCCTGTTGTCAACTCTTAGATTACATCCCCCATTTAATAAGCAATCCTG BF211 BKD_F+FabH2_R For amplifying B. subtilis BKD operon from genomic DNA. Used to create BF211 construct. GACAACAGGAGGAAGAAAGGATGGCAACTGAGTATGACGTAGTC BF211 BKD_R+pET28D For amplifying B. subtilis BKD operon from genomic DNA. Used to create BF211 construct. GCGGTGGCAGCAGCCTAGGTTAATTTAGTAAACAGATGTCTTCTCGTCAATCG BF311 FatB1_R+FabH2_F For amplifying C. viscosissima FatB1 from synthesized DNA fragment. Used to create BF311 construct. AATACCCCCTATGAGTTGTTAATTAATTAAGAAACAGAGTTACCGTTAGAGGTTTTA VA111 CV2025_F+pET28D For amplifying C. violaceum CV2025 from synthesized DNA fragment. Used to create VA111 construct. ACCATCATCACCACAGCCAGGATCCAATAAGGAGGCGAAAATGCAAAAACAAAGGACAACTTCACAG VA111 CV2025_R+pET28D For amplifying C. violaceum CV2025 from synthesized DNA fragment. Used to create VA111 construct. GGCCGCAAGCTTGTCGACCTGCAGGTTAGGCCAGACCGCGAGCTT 73 VA211 pAMT_F+pET28D For amplifying C. chinense pAMT from synthesized DNA fragment. Used to create VA211 construct. CACCATCATCACCACAGCCAGGATCCAATAAGGAGGCGAAAATGGCTAATATCACCAACG VA211 pAMT_R+pET28D For amplifying C. chinense pAMT from synthesized DNA fragment. Used to create VA211 construct. ATGCGGCCGCAAGCTTGTCGACCTGCAGGTCACTGCTTCTGAGATTTCAG ACS111 ACS_F+p28D For amplifying C. chinense ACS from synthesized DNA fragment. Used to create ACS111 construct. CCATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGAT ACS111 ACS_R+p28D For amplifying C. chinense ACS from synthesized DNA fragment. Used to create ACS111 construct. ATGCGGCCGCAAGCTTGTCGACCTGCAGGCGCGGGCGCGCCTTACTTGGTACCTTTGTAC ACS211 FadD_F+p28D For amplifying E. coli FadD from genomic DNA. Used to create ACS211 and ASC212 constructs. CATCATCACCACAGCCAGGATCCTTAAGGAGGTATTTTATGAAGAAGGTTTGGCTTAACC ACS211 FadD_R+p28D For amplifying E. coli FadD from genomic DNA. Used to create ACS211 and ACS212 constructs. CATTATGCGGCCGCAAGCTTGTCGACCTGCAGGTCAGGCTTTATTGTCCACTTTGCC ecFadD_F (V451A) For generating V451A mutation in FadD using PCR site directed mutagenesis. Used to create ACS212 construct. TTCCTGCGCATTGCGGATCGTAAAAAAGACATG ecFadD_R (V451A) For generating V451A mutation in FadD using PCR site directed mutagenesis. Used to create ACS212 construct. ACGATCCGCAATGCGCAGGAATCCTTCTTC ACS311 FadD6_F+p28D For amplifying M. tuberculosis FadD6 from genomic DNA. Used to create ACS311 construct. CATCATCACCACAGCCAGGATCCGTAAGGCAGGTTTTTATGTCCGATTACTACGGCGGC ACS311 FadD6_R+p28D For amplifying M. tuberculosis FadD6 from genomic DNA. Used to create ACS311 construct. CS1111 AT3_F+pET28D For amplifying C. chinense AT3 from synthesized DNA fragment. Used to create CS1111 construct. GCGATCGCTGACGTCGGTACCCTCGAGATAAGGAGGTTTTTTATGGCTTTCGCTCTGCCGTCTTCTCTGGTTTCTGTTTGCGAC CS1111 AT3_R+pET28D For amplifying C. chinense AT3 from synthesized DNA fragment. Used to create CS1111 and CS1112 constructs. GCGGTGGCAGCAGCCTAGGATCTTAATTAATTAAGCGATGAATTCCAGCAGTTCTTCG CS1112 SUMO_F+pET28D For amplifying SUMO tag from pCIOX (Addgene plasmid #51300). Used to create CS1112 construct. GCGATCGCTGACGTCGGTACCCTCGAGATAAGGAGGTTTTTTATGTCGGACTCAG CS1112 SUMO_R+AT3_F For amplifying SUMO tag from pCIOX (Addgene plasmid #51300). Used to create CS1112 construct. GGCAGAGCGAAAGCCATACCACCAATCTGTTCTCTGTGAGCC CS2112 AT3_F+SUMO_R For amplifying C. chinense AT3 from synthesized DNA fragment. Used to create CS1112 construct. GGTGGTATGGCTTTCGCTCTGCCGTCTTCTCTGGTTTCTGTTTGCGACAAATCTTTC CS2112 AT3_R+CV2025_F For amplifying C. chinense AT3 from synthesized DNA fragment. Used to create CS1112 construct. TTCGCCTCCTTATTCCTGCAGGTTAAGCGATGAATTCCAGCAGTTCTTCGTTACGTTCG CS2112 CV2025_F+AT3_R For amplifying C. violaceum CV2025 from synthesized DNA fragment. Used to create CS2112 and CS2212 constructs. CCTGCAGGAATAAGGAGGCGAAAATGCAAAAACAAAGGACAACTTCACAG CS2112 CV2025_R+pET28D For amplifying C. violaceum CV2025 from synthesized DNA fragment. Used to create CS2112 and CS2212 construct. GCATTATGCGGCCGCAAGCTTGTCGACCTGCATTAGGCCAGACCGCGAGCTTTC CS2112 FadD_F+AcTesA_R For amplifying E. coli FadD from genomic DNA. Used to create CS2112 construct. TTAATTAACAACTCATAGGGGGTATTTGTTATGAAGAAGGTTTGGCTTAACC CS2112 FadD_R+pET28D For amplifying E. coli FadD from genomic DNA. Used to create CS2112 construct. CAGCGGTGGCAGCAGCCTAGGATCTTAATTCAGGCTTTATTGTCCACTTTGCC CS2212 MtFadD6_F+pET28D For amplifying M. tuberculosis FadD6 from genomic DNA. Used to create CS2212 construct. ATTAATTAACAACTCATAGGGGGTATTTGTTATGTCCGATTACTACGGCGGCGCACACAC CS2212 MtFadD6_R+pET28D For amplifying M. tuberculosis FadD6 from genomic DNA. Used to create CS2212 construct. ATTGCTCAGCGGTGGCAGCAGCCTAGGATCTTAATCTAGCCCTGCGGTCGCCTTCCGAGC 74 Supplementary Table 2. Sequencing and Colony PCR Primers Primer Sequence Seq_f1_ori_R GGCCCACTACGTGAACCAT Seq_pET28D_I CTACCGGAAGCAGTGTGACC Seq_pET28D_II ACCCCGCCAGCCTAGCCGG Seq_pET28D_III GACCGATGCCCTTGAGAGCC Seq_pET28D_IV CGATGCGTCCGGCGTAGAGG Seq_TesA_F CATCAGCCGCGTTGGGTG Seq_TesA_R CGGAACATCAAACTCTTTGGCG Seq_AcTesA_F ACTGCGCGGTCAACCTCC Seq_AcTesA_R GATTCGATTGAATCATCTGCGGAGG Seq_FatB1_F CTGCCGGACTGGTCTATGCTG Seq_FatB1_R ACCAACAGAGTCAACCAGCA Seq_FabH2_R GAAATATTCCTGTACGCGGC Seq_FabH2_F CTTGTCGGTTCCGGAAAAAT Seq_IpdV_R ACAACGGCTGTTTTTAAGCC Seq_IpdV_R_II GAGCTAACTGCAGGCCACCG Seq_BkdAA_R_II GTGCTGCTTCCTGTCCTTGACAAG Seq_BkdAA_R_I CTGTCATCGTCATCACTGGAATGTGG Seq_BkdAB_R_I TGAGAATGATACAGGGCTCCGTG Seq_BkdB_R_III CGTCATTTGTTCAATTGCCATGATTGTATCCTC Seq_BkdB_R_II TGCTTTTCTTACACCTGTGACAGGG Seq_ACS_F_I GGTTACTACAAACGTGAAGACCTGACC Seq_ACS_R_I GTACTTAGAAGCGTTCGGGAAGG Seq_ACS_R_II AGGAAACCACCAGCAGCG Seq_ACS_R_III CGGTCGATGATCTTCAGAGAACC Seq_FadD_F GCGCTGCCGCTGTATCAC Seq_FadD_R CAAGGTGTTAACGCCCGTGATAG Seq_MtFadD6_F GACGGCGTTCGTCTACATCGG Seq_MtFadD6_R GGGTGGTGAACTCATCCCAGATC Seq_CV2025_F CGGATAAAGTTGCGGCGTTCG Seq_CV2025_R CAGCAGCACATCGTACTTACGG Seq_pAMT_F_II GAATGCCCGCACTACTGGGC Seq_pAMT_R GAAAGCCGCAACGGTTTCCGG Seq_AT3_F_I TGCTGCAGACCTCTCTGTCTCAGAC Seq_AT3_R_I GTGCGGGTGGTCCAGGATTTC 75 Supplementary Table 3. Primers Used for BF211 Twin Primer Assembly Primer Sequence AcTesA_F+pET28D TPAL ACGTCGGTACCCTCGAGAGTAAGGAGCTTATTATATGGGCAAAACCATTCTGATTCTG AcTesA_F+pET28D TPAS AGTAAGGAGCTTATTATATGGGCAAAACCATTCTGATTCTG AcTesA_R+FabH2_F ATACCCCCTATGAGTTGTTAATTAATTACAGCGCGCCTTTGATATACG AcTesA_R+FabH2_F TPAS TTACAGCGCGCCTTTGATATACG BKD_F+FabH2_R TPAL TGACAACAGGAGGAAGAAAGGATGGCAACTGAGTATGACGTAGTC BKD_F+FabH2_R TPAS ATGGCAACTGAGTATGACGTAGTC BKD_R+pET28-D TPAL TTAGTAAACAGATGTCTTCTCGTC BKD_R+pET28-D TPAS AATCGATTCTAAAATTTGTTTCACTCGTC FabH2_F+Thioes_R TTAATTAACAACTCATAGGGGGTATTTGTTATGTCAAAAGCAAAAATTACAGCTATCGGC FabH2_F+Thioes_R TPAS TTGTTATGTCAAAAGCAAAAATTACAGCTATCGGC FabH2_R+BKD_F CCTTTCTTCCTCCTGTTGTCAACTCTTAGATTACATCCCCCATTTAATAAGCAATCCTG FabH2_R+BKD_F TPAS ACTCTTAGATTACATCCCCCATTTAATAAGCAATCCTG pET28-D PacI ATTAACCTAGGCTGCTGCCACC pET28-D XhoI TPAL CTCGAGGGTACCGACGT pET28-D XhoI TPAS CAGCGATCGCGTGGCCGGCCGA pET28-D+BKD_R TPAL GACGAGAAGACATCTGTTTACTAAATTAACCTAGGCTGCTGCCACC 76 Supplementary Table 4. Plasmids and Bacterial Strains Used in this Study Plasmid Relevant Features Targeted Outcome pET28D Modified pET28a(+) with two MCSs Backbone vector of all the assembled constructs Chapter 2 SF111 pET28D, PT7-ecTesA’; Plac-FadR C12-C18 SCFA SF112 pET28D, PT7-ecTesA’ Y145K; Plac-FadR C6-C16 SCFA SF211 pET28D, PT7-abAcTesA’; Plac-FadR C6-C18 SCFA SF311 pET28D, PT7-cv1FatB1; Plac-FadR C8-C10 SCFA BF111 pET28D, PT7-ecTesA’-bsFabH2-bsBKD; Plac-FadR C12-C18 BCFA BF112 pET28D, PT7-ecTesA’ Y145K-bsFabH2-bsBKD; Plac-FadR C6-C16 BCFA BF211 pET28D, PT7-abAcTesA’-bsFabH2-bsBKD; Plac-FadR C6-C18 BCFA BF311 pET28D, PT7-cv1FatB1-bsFabH2-bsBKD; Plac-FadR C8-C10 BCFA Chapter 3 VA111 pET28D, PT7-cv2CV2025 Vanillylamine production VA211 pET28D, PT7-ccpAMT Vanillylamine production Chapter 4 ACS111 pET28D, PT7-ccASC CoA activation of BCFA ACS211 pET28D, PT7-ecFadD CoA activation of >=C10 fatty acids ACS212 pET28D, PT7-ecFadD V451A CoA activation of >=C8 fatty acids ACS411 pET28D, PT7-mtbFadD6 CoA activation of >=C6 fatty acids Chapter 5 CS1111 pET28D, PT7-ccAT3 Analyze protein solubility without the SUMO tag CS1112 pET28D, PT7-ccSUMO-AT3 Analyze protein solubility with the SUMO tag CS2112 pET28D, PT7-abAcTesA’-ecFadD-bs; PT7-ccSUMO-AT3-cv2CV2025; Plac-FadR >=C10 Capsaicinoid synthesis CS2212 pET28D, PT7-abAcTesA’-mtbFadD6; PT7-ccSUMO-AT3-cv2CV2025; Plac-FadR >=C6 Capsaicinoid synthesis Strain Relevant Features TransGen Trans1-T1 F-φ80(lacZ)ΔM15ΔlacX74hsdR(rk-, mk+)ΔrecA1398endA1tonA BL21(DE3) huA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 NEB T7 Express LysY MiniF lysY lacIq(CamR) / fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) endA1 Δ(mcrC-mrr) 114::IS10 77 Supplementary Table 5. GenBank Accession Numbers and Sequences Fragment GenBank Accession No. Sequence E. coli TesA’ (leaderless) AAA24664.1 ATGGCGGACACGTTATTGATTCTGGGTGATAGCCTGAGCGCCGGGTATCGAATGTCTGCCAGCGCGGCCTGGCCTGCCTTGTTGAATGATAAGTGGCAGAGTAAAACGTCGGTAGTTAATGCCAGCATCAGCGGCGACACCTCGCAACAAGGACTGGCGCGCCTTCCGGCTCTGCTGAAACAGCATCAGCCGCGTTGGGTGCTGGTTGAACTGGGCGGCAATGACGGTTTGCGTGGTTTTCAGCCACAGCAAACCGAGCAAACGCTGCGCCAGATTTTGCAGGATGTCAAAGCCGCCAACGCTGAACCATTGTTAATGCAAATACGTCTGCCTGCAAACTATGGTCGCCGTTATAATGAAGCCTTTAGCGCCATTTACCCCAAACTCGCCAAAGAGTTTGATGTTCCGCTGCTGCCCTTTTTTATGGAAGAGGTCTACCTCAAGCCACAATGGATGCAGGATGACGGTATTCATCCCAACCGCGACGCCCAGCCGTTTATTGCCGACTGGATGGCGAAGCAGTTGCAGCCTTTAGTAAATCATGACTCATAA A. bayly AcTesA’ (leaderless) CAG67945.1 ATGGGCAAAACCATTCTGATTCTGGGCGACAGCCTGAGCGCTGGCTATGGCATTAATCCGGAGCAAGGCTGGGTTGCGTTGTTGCAAAAACGTCTGGATCAGCAGTTTCCGAAACAACATAAAGTTATTAATGCAAGTGTTAGCGGTGAGACTACTAGTGGCGCGCTGGCTCGACTGCCGAAGCTGTTGACCACTTACCGCCCGAATGTGGTTGTTATTGAACTGGGTGGCAACGATGCACTGCGCGGTCAACCTCCGCAGATGATTCAATCGAATCTGGAAAAACTGATTCAACATTCGCAAAAGGCGAAATCTAAAGTGGTTGTGTTTGGCATGAAAATACCCCCTAATTATGGCACTGCTTATTCTCAAGCGTTTGAAAATAATTATAAAGTTGTAAGTCAAACTTACCAGGTGAAACTGCTGCCGTTTTTTCTGGATGGCGTGGCGGGCCACAAAAGTTTGATGCAGAACGATCAGATTCACCCGAATGCGAAGGCGCAAAGTATCCTGCTGAATAATGCGTACCCGTATATCAAAGGCGCGCTGTAA C. viscosissima FatB1 AEM72522.1 ATGGTTGCTGCTGCTGCTACCTCTGCTTTCTTCCCGGTTCCGGCTCCGGGTACCTCTCCGAAACCGGGTAAATCTGGTAACTGGCCGTCTTCTCTGTCTCCGACCTTCAAACCGAAATCTATCCCGAACGGTGGTTTCCAGGTTAAAGCTAACGCTTCTGCTCACCCGAAAGCTAACGGTTCTGCTGTTAACCTGAAATCTGGTTCTCTGAACACCCAGGAAGACACCTCTTCTTCTCCGCCGCCGCGTGCTTTCCTGAACCAGCTGCCGGACTGGTCTATGCTGCTGACCGCTATCACCACCGTTTTCGTTGCTGCTGAAAAACAGTGGACCATGCTGGACCGTAAATCTAAACGTCCGGACATGCTGGTTGACTCTGTTGGTCTGAAATCTATCGTTCGTGACGGTCTGGTTTCTCGTCACTCTTTCTCTATCCGTTCTTACGAAATCGGTGCTGACCGTACCGCTTCTATCGAAACCCTGATGAACCACCTGCAGGAAACCACCATCAACCACTGCAAATCTCTGGGTCTGCACAACGACGGTTTCGGTCGTACCCCGGGTATGTGCAAAAACGACCTGATCTGGGTTCTGACCAAAATGCAGATCATGGTTAACCGTTACCCGACCTGGGGTGACACCGTTGAAATCAACACCTGGTTCTCTCAGTCTGGTAAAATCGGTATGGCTTCTGACTGGCTGATCTCTGACTGCAACACCGGTGAAATCCTGATCCGTGCTACCTCTGTTTGGGCTATGATGAACCAGAAAACCCGTCGTTTCTCTCGTCTGCCGTACGAAGTTCGTCAGGAACTGACCCCGCACTTCGTTGACTCTCCGCACGTTATCGAAGACAACGACCAGAAACTGCGTAAATTCGACGTTAAAACCGGTGACTCTATCCGTAAAGGTCTGACCCCGCGTTGGAACGACCTGGACGTTAACCAGCACGTTTCTAACGTTAAATACATCGGTTGGATCCTGGAATCTATGCCGATCGAAGTTCTGGAAACCCAGGAACTGTGCTCTCTGACCGTTGAATACCGTCGTGAATGCGGTATGGACTCTGTTCTGGAATCTGTTACCGCTGTTGACCCGTCTGAAAACGGTGGTCGTTCTCAGTACAAACACCTGCTGCGTCTGGAAGACGGTACCGACATCGTTAAATCTCGTACCGAATGGCGTCCGAAAAACGCTGGTACCAACGGTGCTATCTCTACCTCTACCGCTAAAACCTCTAACGGTAACTCTGTTTCTTAA E. coli FadR ACT29461.1 ATGGTCATTAAGGCGCAAAGCCCGGCGGGTTTCGCGGAAGAGTACATTATTGAAAGTATCTGGAATAACCGCTTCCCTCCCGGGACTATTTTGCCCGCAGAACGTGAACTTTCAGAATTAATTGGCGTAACGCGTACTACGTTACGTGAAGTGTTACAGCGTCTGGCACGAGATGGCTGGTTGACCATTCAACATGGCAAGCCGACGAAGGTGAATAATTTCTGGGAAACTTCCGGTTTAAATATCCTTGAAACACTGGCGCGACTGGATCACGAAAGTGTGCCGCAGCTTATTGATAATTTGCTGTCGGTGCGTACCAATATTTCCACTATTTTTATTCGCACCGCGTTTCGTCAGCATCCCGATAAAGCGCAGGAAGTGCTGGCTACCGCTAATGAAGTGGCCGATCACGCCGATGCCTTTGCCGAGCTGGATTACAACATATTCCGCGGCCTGGCGTTTGCTTCCGGCAACCCGATTTACGGTCTGATTCTTAACGGGATGAAAGGGCTGTATACGCGTATTGGTCGTCACTATTTCGCCAATCCGGAAGCGCGCAGTCTGGCGCTGGGCTTCTACCACAAACTGTCGGCGTTGTGCAGTGAAGGCGCGCACGATCAGGTGTACGAAACAGTGCGTCGCTATGGGCATGAGAGTGGCGAGATTTGGCACCGGATGCAGAAAAATCTGCCGGGTGATTTAGCCATTCAGGGGCGATAA B. subtilis FabH2 CAB12857.1 ATGTCAAAAGCAAAAATTACAGCTATCGGCACCTATGCGCCGAGCAGACGTTTAACCAATGCAGATTTAGAAAAGATCGTTGATACCTCTGATGAATGGATCGTTCAGCGCACAGGAATGAGAGAACGCCGGATTGCGGATGAACATCAATTTACCTCTGATTTATGCATAGAAGCGGTGAAGAATCTCAAGAGCCGTTATAAAGGAACGCTTGATGATGTCGATATGATCCTCGTTGCCACAACCACATCCGATTACGCCTTTCCGAGTACGGCATGCCGCGTACAGGAATATTTCGGCTGGGAAAGCACCGGCGCGCTGGATATTAATGCGACATGCGCCGGGCTGACATACGGCCTCCATTTGGCAAATGGATTGATCACATCTGGCCTTCATCAAAAAATTCTCGTCATCGCCGGAGAGACGTTATCAAAGGTAACCGATTATACCGATCGAACGACATGCGTACTGTTCGGCGATGCCGCGGGTGCGCTGTTAGTAGAACGAGATGAAGAGACGCCGGGATTTCTTGCGTCTGTACAAGGAACAAGCGGGAACGGCGGCGATATTTTGTATCGTGCCGGACTGCGAAATGAAATAAACGGTGTGCAGCTTGTCGGTTCCGGAAAAATGGTGCAAAACGGACGCGAGGTATATAAATGGGCCGCAAGAACCGTCCCTGGCGAATTTGAACGGCTTTTACATAAAGCAGGACTCAGCTCCGATGATCTCGATTGGTTTGTTCCTCACAGCGCCAA78 CTTGCGCATGATCGAGTCAATTTGTGAAAAAACACCGTTCCCGATTGAAAAAACGCTCACTAGTGTTGAGCACTACGGAAACACGTCTTCGGTTTCAATTGTTTTGGCGCTCGATCTCGCAGTGAAAGCCGGGAAGCTGAAAAAAGATCAAATCGTTTTGCTTTTCGGGTTTGGCGGCGGATTAACCTATACAGGATTGCTTATTAAATGGGGGATGTAA B. subtilis BKD operon BAA12597.1 AAA22278.1 AAA22279.1 AAA22280.1 ATGGCAACTGAGTATGACGTAGTCATTCTGGGCGGCGGTACCGGCGGTTATGTTGCGGCCATCAGAGCCGCTCAGCTCGGCTTAAAAACAGCCGTTGTGGAAAAGGAAAAACTCGGGGGAACATGTCTGCATAAAGGCTGTATCCCGAGTAAAGCGCTGCTTAGAAGCGCAGAGGTATACCGGACAGCTCGTGAAGCCGATCAATTCGGAGTGGAAACGGCTGGCGTGTCCCTCAACTTTGAAAAAGTGCAGCAGCGTAAGCAAGCCGTTGTTGATAAGCTTGCAGCGGGTGTAAATCATTTAATGAAAAAAGGAAAAATTGACGTGTACACCGGATATGGACGTATCCTTGGACCGTCAATCTTCTCTCCGCTGCCGGGAACAATTTCTGTTGAGCGGGGAAATGGCGAAGAAAATGACATGCTGATCCCGAAACAAGTGATCATTGCAACAGGATCAAGACCGAGAATGCTTCCGGGTCTTGAAGTGGACGGTAAGTCTGTACTGACTTCAGATGAGGCGCTCCAAATGGAGGAGCTGCCACAGTCAATCATCATTGTCGGCGGAGGGGTTATCGGTATCGAATGGGCGTCTATGCTTCATGATTTTGGCGTTAAGGTAACGGTTATTGAATACGCGGATCGCATATTGCCGACTGAAGATCTAGAGATTTCAAAAGAAATGGAAAGTCTTCTTAAGAAAAAAGGCATCCAGTTCATAACAGGGGCAAAAGTGCTGCCTGACACAATGACAAAAACATCAGACGATATCAGCATACAAGCGGAAAAAGACGGAGAAACCGTTACCTATTCTGCTGAGAAAATGCTTGTTTCCATCGGCAGACAGGCAAATATCGAAGGCATCGGCCTAGAGAACACCGATATTGTTACTGAAAATGGCATGATTTCAGTCAATGAAAGCTGCCAAACGAAGGAATCTCATATTTATGCAATCGGAGACGTAATCGGTGGCCTGCAGTTAGCTCACGTTGCTTCACATGAGGGAATTATTGCTGTTGAGCATTTTGCAGGTCTCAATCCGCATCCGCTTGATCCGACGCTTGTGCCGAAGTGCATTTACTCAAGCCCTGAAGCTGCCAGTGTCGGCTTAACCGAAGACGAAGCAAAGGCGAACGGGCATAATGTCAAAATCGGCAAGTTCCCATTTATGGCGATTGGAAAAGCGCTTGTATACGGTGAAAGCGACGGTTTTGTCAAAATCGTGGCTGACCGAGATACAGATGATATTCTCGGCGTTCATATGATTGGCCCGCATGTCACCGACATGATTTCTGAAGCGGGTCTTGCCAAAGTGCTGGACGCAACACCGTGGGAGGTCGGGCAAACGATTCACCCGCATCCAACGCTTTCTGAAGCAATTGGAGAAGCTGCGCTTGCCGCAGATGGCAAAGCCATTCATTTTTAAAAGCATAAAGGAGGGGCTTGAATGAGTACAAACCGACATCAAGCACTAGGGCTGACTGATCAGGAAGCCGTTGATATGTATAGAACCATGCTGTTAGCAAGAAAAATCGATGAAAGAATGTGGCTGTTAAACCGTTCTGGCAAAATTCCATTTGTAATCTCTTGTCAAGGACAGGAAGCAGCACAGGTAGGAGCGGCTTTCGCACTTGACCGTGAAATGGATTATGTATTGCCGTACTACAGAGACATGGGTGTCGTGCTCGCGTTTGGCATGACAGCAAAGGACTTAATGATGTCCGGGTTTGCAAAAGCAGCAGATCCGAACTCAGGAGGCCGCCAGATGCCGGGACATTTCGGACAAAAGAAAAACCGCATTGTGACGGGATCATCTCCGGTTACAACGCAAGTGCCGCACGCAGTCGGTATTGCGCTTGCGGGACGTATGGAGAAAAAGGATATCGCAGCCTTTGTTACATTCGGGGAAGGGTCTTCAAACCAAGGCGATTTCCATGAAGGGGCAAACTTTGCCGCTGTCCATAAGCTGCCGGTTATTTTCATGTGTGAAAACAACAAATACGCAATCTCAGTGCCTTACGATAAGCAAGTCGCATGTGAGAACATTTCCGACCGTGCCATAGGCTATGGGATGCCTGGCGTAACTGTGAATGGAAATGATCCGCTGGAAGTTTATCAAGCGGTTAAAGAAGCACGCGAAAGGGCACGCAGAGGAGAAGGCCCGACATTAATTGAAACGATTTCTTACCGCCTTACACCACATTCCAGTGATGACGATGACAGCAGCTACAGAGGCCGTGAAGAAGTAGAGGAAGCGAAAAAAAGTGATCCCCTGCTTACTTATCAAGCTTACTTAAAGGAAACAGGCCTGCTGTCCGATGAGATAGAACAAACCATGCTGGATGAAATTATGGCAATCGTAAATGAAGCGACGGATGAAGCGGAGAACGCCCCATATGCAGCTCCTGAGTCAGCGCTTGATTATGTTTATGCGAAGTAGGGAGGAAGAACAAATGTCAGTAATGTCATATATTGATGCAATCAATTTGGCGATGAAAGAAGAAATGGAACGAGATTCTCGCGTTTTCGTCCTTGGGGAAGATGTAGGAAGAAAAGGCGGTGTGTTTAAAGCGACAGCGGGACTCTATGAACAATTTGGGGAAGAGCGCGTTATGGATACGCCGCTTGCTGAATCTGCAATCGCAGGAGTCGGTATCGGAGCGGCAATGTACGGAATGAGACCGATTGCTGAAATGCAGTTTGCTGATTTCATTATGCCGGCAGTCAACCAAATTATTTCTGAAGCGGCTAAAATCCGCTACCGCAGCAACAATGACTGGAGCTGTCCGATTGTCGTCAGAGCGCCATACGGCGGAGGCGTGCACGGAGCCCTGTATCATTCTCAATCAGTCGAAGCAATTTTCGCCAACCAGCCCGGACTGAAAATTGTCATGCCATCAACACCATATGACGCGAAAGGGCTCTTAAAAGCCGCAGTTCGTGACGAAGACCCCGTGCTGTTTTTTGAGCACAAGCGGGCATACCGTCTGATAAAGGGCGAGGTTCCGGCTGATGATTATGTCCTGCCAATCGGCAAGGCGGACGTAAAAAGGGAAGGCGACGACATCACAGTGATCACATACGGCCTGTGTGTCCACTTCGCCTTACAAGCTGCAGAACGTCTCGAAAAAGATGGCATTTCAGCGCATGTGGTGGATTTAAGAACAGTTTACCCGCTTGATAAAGAAGCCATCATCGAAGCTGCGTCCAAAACTGGAAAGGTTCTTTTGGTCACAGAAGATACAAAAGAAGGCAGCATCATGAGCGAAGTAGCCGCAATTATATCCGAGCATTGTCTGTTCGACTTAGACGCGCCGATCAAACGGCTTGCAGGTCCTGATATTCCGGCTATGCCTTATGCGCCGACAATGGAAAAATACTTTATGGTCAACCCTGATAAAGTGGAAGCGGCGATGAGAGAATTAGCGGAGTTTTAAAGACGTAAGGGAGGATACAATCATGGCAATTGAACAAATGACGATGCCGCAGCTTGGAGAAAGCGTAACAGAGGGGACGATCAGCAAATGGCTTGTCGCCCCCGGTGATAAAGTGAACAAATACGATCCGATCGCGGAAGTCATGACAGATAAGGTAAATGCAGAGGTTCCGTCTTCTTTTACTGGTACGATAACAGAGCTTGTGGGAGAAGAAGGCCAAACCCTGCAAGTCGGAGAAATGATTTGCAAAATTGAAACAGAAGGCGCGAATCCGGCTGAACAAAAACAAGAACAGCCAGCAGCATCAGAAGCCGCTGAGAACCCTGTTGCAAAAAGTGCTGGAGCAGCCGATCAGCCCAATAAAAAGCGCTACTCGCCAGCTGTTCTCCGTTTGGCCGGAGAGCACGGCATTGACCTCGATCAAGTGACAGGAACTGGTGCCGGCGGGCGCATCACACGAAAAGATATTCAGCG79 CTTAATTGAAACAGGCGGCGTGCAAGAACAGAATCCTGAGGAGCTGAAAACAGCAGCTCCTGCACCGAAGTCTGCATCAAAACCTGAGCCAAAAGAAGAGACGTCATATCCTGCGTCTGCAGCCGGTGATAAAGAAATCCCTGTCACAGGTGTAAGAAAAGCAATTGCTTCCAATATGAAGCGAAGCAAAACAGAAATTCCGCATGCTTGGACGATGATGGAAGTCGACGTCACAAATATGGTTGCATATCGCAACAGTATAAAAGATTCTTTTAAGAAGACAGAAGGCTTTAATTTAACGTTCTTCGCCTTTTTTGTAAAAGCGGTCGCTCAGGCGTTAAAAGAATTCCCGCAAATGAATAGCATGTGGGCGGGGGACAAAATTATTCAGAAAAAGGATATCAATATTTCAATTGCAGTTGCCACAGAGGATTCTTTATTTGTTCCGGTGATTAAAAACGCTGATGAAAAAACAATTAAAGGCATTGCGAAAGACATTACCGGCCTAGCTAAAAAAGTAAGAGACGGAAAACTCACTGCAGATGACATGCAGGGAGGCACGTTTACCGTCAACAACACAGGTTCGTTCGGGTCTGTTCAGTCGATGGGCATTATCAACTACCCTCAGGCTGCGATTCTTCAAGTAGAATCCATCGTCAAACGCCCGGTTGTCATGGACAATGGCATGATTGCTGTCAGAGACATGGTTAATCTGTGCCTGTCATTAGATCACAGAGTGCTTGACGGTCTCGTGTGCGGACGATTCCTCGGACGAGTGAAACAAATTTTAGAATCGATTGACGAGAAGACATCTGTTTACTAA C. annum ACS ACF17663.1 ATGGCTACCGACAAATTCATCATTGAAGTTGAATCTGCTAAACCGGCTAAAGACGGTCGTCCGTCTATGGGTCCGGTTTACCGTTCTATCTTCGCTAAACACGGTTTCCCGCCGCCGATCCCTGGGCTGGATTCTTGCTGGGACATCTTCCGTATGTCGGTTGAAAAGTACCCGAACAACCGTATGCTGGGTCGTCGTGAAATCGTTGACGGTAAACCGGGTAAATACGTTTGGATGTCTTACAAAGAAGTTTACGACATCGTTATCAAAGTGGGTAACTCTATCCGTTCTATCGGTGTTGACGTTGGTGACAAATGCGGTATCTACGGTGCTAACTGCCCGGAATGGATCATCTCTATGGAAGCTTGCAACGCTCACGGTCTGTACTGCGTTCCGCTGTACGACACCCTGGGTGCTGGTGCTGTTGAATTCATCATCTCTCACGCTGAAGTTACCATCGCTTTCGTTGAAGAAAAAAAACTGCCGGAACTGCTGAAAACCTTCCCGAACGCTTCTAAGTACCTGAAAACCATCGTTTCTTTCGGTAAAGTTACCCCGGAACAGAAAAAAGAACTGGAAGAATTCGGTGTTGTTCTGTACTCTTGGGACGAATTCCTGCAGCTGGGTTCTGGTAAACAGTTCGACCTGCCGGTTAAAAAGAAGGAAGACATCTGCACCATCATGTACACCTCTGGTACCACCGGTGACCCGAAAGGTGTTCTGATCTCTAACACCTCTATCGTTACCCTGATCGCTGGTGTTCGTCGTTTCCTGGGTTCTGTTGACGAATCTCTGAACGTTGACGACGTTTACCTGTCTTACCTGCCGCTGGCTCACATCTTCGACCGTGTTATTGAAGAATGCTTCATCCACCACGGTGCTTCTATCGGTTTCTGGCGTGGTGACGTTAAACTGCTGACCGAGGACATCGGTGAACTGAAACCGACCGTCTTCTGCGCTGTTCCGCGTGTTCTGGACCGTATCTACTCTGGTCTGCAGCAGAAGATCGCTGCTGGTGGTTTCCTGAAATCTACCCTGTTCAACCTGGCTTACGCTTACAAACACCACAACCTGAAAAAAGGTCGTAAACACTTCGAAGCTTCTCCGCTGTCTGACAAAGTCGTTTTTTCTAAAGTTAAAGAAGGTCTGGGTGGTCGTGTTCGTCTGATCTTGTCTGGTGCGGCTCCTCTGGCGGCGCATGTTGAAGCTTTCCTGCGTGTTGTTGCTTGCTGCCACGTTCTGCAGGGTTACGGTCTGACAGAAACCTGCGCTGGTACCTTCGTTTCTCTGCCGAACCGTTACGACATGCTGGGTACCGTTGGTCCGCCGGTTCCGAACGTTGACGTTTGCCTGGAATCTGTTCCGGAAATGTCTTACGACGCTCTGTCTTCTACCCCGCGTGGTGAAGTTTGCGTTCGTGGTGACATCCTGTTCTCTGGTTACTACAAACGTGAAGACCTGACCAAAGAAGTTATGATCGACGGTTGGTTCCACACCGGTGATGTTGGTGAATGGCAGCCGAACGGTTCTCTGAAGATCATCGACCGTAAAAAGAATATTTTTAAACTGTCTCAGGGTGAATACGTTGCAGTGGAAAACCTGGAAAACATCTACGGTAACAACCCGATCATCGACTCTATCTGGATCTACGGTAACTCTTTCGAATCTTTCCTGGTTGCCGTTATCAACCCGAACCAGCGTGCTGTTGAACAGTGGGCTGAAGTTAACGGTCTGTCTGGTGACTTCGCTTCTCTGTGCGAAAAACCGGAAGTTAAGGAATACATCCTGCGTGAACTGACCAAAACGGGTAAGGAAAAAAAACTGAAAGGTTTCGAATTCCTGAAAGCTGTTCACCTGGACCCGGTTCCGTTCGACATGGAACGTGACCTGCTGACCCCGACCTTCAAAAAAAAACGTCCGCAGCTGCTGAAATACTACAAAGACGTTATCGACTCTATGTACAAAGGTACCAAGTAA E. coli FadD CAA50321.1 ATGAAGAAGGTTTGGCTTAACCGTTATCCCGCGGACGTTCCGACGGAGATCAACCCTGACCGTTATCAATCTCTGGTAGATATGTTTGAGCAGTCGGTCGCGCGCTACGCCGATCAACCTGCGTTTGTGAATATGGGGGAGGTAATGACCTTCCGCAAGCTGGAAGAACGCAGTCGCGCGTTTGCCGCTTATTTGCAACAAGGGTTGGGGCTGAAGAAAGGCGATCGCGTTGCGTTGATGATGCCTAATTTATTGCAATATCCGGTGGCGCTGTTTGGCATTTTGCGTGCCGGGATGATCGTCGTAAACGTTAACCCGTTGTATACCCCGCGTGAGCTTGAGCATCAGCTTAACGATAGCGGCGCATCGGCGATTGTTATCGTGTCTAACTTTGCTCACACACTGGAAAAAGTGGTTGATAAAACCGCCGTTCAGCACGTAATTCTGACCCGTATGGGCGATCAGCTATCTACGGCAAAAGGCACGGTAGTCAATTTCGTTGTTAAATACATCAAGCGTTTGGTGCCGAAATACCATCTGCCAGATGCCATTTCATTTCGTAGCGCACTGCATAACGGCTACCGGATGCAGTACGTCAAACCCGAACTGGTGCCGGAAGATTTAGCTTTTCTGCAATACACCGGCGGCACCACTGGTGTGGCGAAAGGCGCGATGCTGACTCACCGCAATATGCTGGCGAACCTGGAACAGGTTAACGCGACCTATGGTCCGCTGTTGCATCCGGGCAAAGAGCTGGTGGTGACGGCGCTGCCGCTGTATCACATTTTTGCCCTGACCATTAACTGCCTGCTGTTTATCGAACTGGGTGGGCAGAACCTGCTTATCACTAACCCGCGCGATATTCCAGGGTTGGTAAAAGAGTTAGCGAAATATCCGTTTACCGCTATCACGGGCGTTAACACCTTGTTCAATGCGTTGCTGAACAATAAAGAGTTCCAGCAGCTGGATTTCTCCAGTCTGCATCTTTCCGCAGGCGGAGGGATGCCAGTGCAGCAAGTGGTGGCAGAGCGTTGGGTGAAACTGACAGGACAGTATCTGCTGGAAGGCTATGGCCTTACCGAGTGTGCGCCGCTGGTCAGCGTTAACCCATATGATATTGATTATCATAGTGGTAGCATCGGTTTGCCGGTGCCGTCGACGGAAGCCAAACTGGTGGATGATGATGATAATGAAGTACCACCGGGTCAACCGGGTGAGCTTTGTGTCAAAGGACCGCAGGTGATGCTGGGTTACTGGCAGCGTCCGGATGCTACAGATGAGATCATCAAAAATGGCTGGTTACACACCGGCGACATCGCGGTGATGGATGAAGAAGGATTCCTGCGCATTGCGGATCGTAAAAAAGACATGATTCTGGTTTCCGGTTTTAACGTCT80 ATCCCAACGAGATTGAAGATGTCGTCATGCAGCATCCTGGCGTACAGGAAGTCGCGGCTGTTGGCGTACCTTCCGGCTCCAGTGGTGAAGCGGTGAAAATCTTCGTAGTGAAAAAAGATCCATCGCTTACCGAAGAGTCACTGGTGACCTTTTGCCGCCGTCAGCTCACGGGCTACAAAGTACCGAAGCTGGTGGAGTTTCGTGATGAGTTACCGAAATCTAACGTCGGAAAAATTTTGCGACGAGAATTACGTGACGAAGCGCGCGGCAAAGTGGACAATAAAGCCTGA M. tuberculosis FadD6 CCP43962.1 ATGTCCGATTACTACGGCGGCGCACACACAACGGTCAGGCTGATCGACCTGGCAACTCGGATGCCGCGAGTGTTGGCGGACACGCCGGTGATTGTGCGTGGGGCAATGACCGGGCTGCTGGCCCGGCCGAATTCCAAGGCGTCGATCGGCACGGTGTTCCAGGACCGGGCCGCTCGCTACGGTGACCGAGTCTTCCTGAAATTCGGCGATCAGCAGCTGACCTACCGCGACGCTAACGCCACCGCCAACCGGTACGCCGCGGTGTTGGCCGCCCGCGGCGTCGGCCCCGGCGACGTCGTTGGCATCATGTTGCGTAACTCACCCAGCACAGTCTTGGCGATGCTGGCCACGGTCAAGTGCGGCGCTATCGCCGGCATGCTCAACTACCACCAGCGCGGCGAGGTGTTGGCGCACAGCCTGGGTCTGCTGGACGCGAAGGTACTGATCGCAGAGTCCGACTTGGTCAGCGCCGTCGCCGAATGCGGCGCCTCGCGCGGCCGGGTAGCGGGCGACGTGCTGACCGTCGAGGACGTGGAGCGATTCGCCACAACGGCGCCCGCCACCAACCCGGCGTCGGCGTCGGCGGTGCAAGCCAAAGACACCGCGTTCTACATCTTCACCTCGGGCACCACCGGATTTCCCAAGGCCAGTGTCATGACGCATCATCGGTGGCTGCGGGCGCTGGCCGTCTTCGGAGGGATGGGGCTGCGGCTGAAGGGTTCCGACACGCTCTACAGCTGCCTGCCGCTGTACCACAACAACGCGTTAACGGTCGCGGTGTCGTCGGTGATCAATTCTGGGGCGACCCTGGCGCTGGGTAAGTCGTTTTCGGCGTCGCGGTTCTGGGATGAGGTGATTGCCAACCGGGCGACGGCGTTCGTCTACATCGGCGAAATCTGCCGTTATCTGCTCAACCAGCCGGCCAAGCCGACCGACCGTGCCCACCAGGTGCGGGTGATCTGCGGTAACGGGCTGCGGCCGGAGATCTGGGATGAGTTCACCACCCGCTTCGGGGTCGCGCGGGTGTGCGAGTTCTACGCCGCCAGCGAAGGCAACTCGGCCTTTATCAACATCTTCAACGTGCCCAGGACCGCCGGGGTATCGCCGATGCCGCTTGCCTTTGTGGAATACGACCTGGACACCGGCGATCCGCTGCGGGATGCGAGCGGGCGAGTGCGTCGGGTACCCGACGGTGAACCCGGCCTGTTGCTTAGCCGGGTCAACCGGCTGCAGCCGTTCGACGGCTACACCGACCCGGTTGCCAGCGAAAAGAAGTTGGTGCGCAACGCTTTTCGAGATGGCGACTGTTGGTTCAACACCGGTGACGTGATGAGCCCGCAGGGCATGGGCCATGCCGCCTTCGTCGATCGGCTGGGCGACACCTTCCGCTGGAAGGGCGAGAATGTCGCCACCACTCAGGTCGAAGCGGCACTGGCCTCCGACCAGACCGTCGAGGAGTGCACGGTCTACGGCGTCCAGATTCCGCGCACCGGCGGGCGCGCCGGAATGGCCGCGATCACACTGCGCGCTGGCGCCGAATTCGACGGCCAGGCGCTGGCCCGAACGGTTTACGGTCACTTGCCCGGCTATGCACTTCCGCTCTTTGTTCGGGTAGTGGGGTCGCTGGCGCACACCACGACGTTCAAGAGTCGCAAGGTGGAGTTGCGCAACCAGGCCTATGGCGCCGACATCGAGGATCCGCTGTACGTACTGGCCGGCCCGGACGAAGGATATGTGCCGTACTACGCCGAATACCCTGAGGAGGTTTCGCTCGGAAGGCGACCGCAGGGCTAG C. chinense pAMT AAC78480.1 ATGGCTAATATCACCAACGAATTCATGGGTCACGACATGCTGGCTCCGTTCACCGCTGGTTGGCAGTCTGACATGGAACCGCTGGTTATTGAAAAATCTGAAGGTTCTTACGTTTACGACATCAACGGTAAAAAATACCTGGACACCCTGTCTGGTCTGTGGTGCGCTACCCTGGGTGGTTCTGAAACCCGTCTGGTTGAAGCTGCTAACAAACAGCTGAACACCCTGCCGTTCTACCACTCTTTCTGGAACCGCACCACCAAACCGTCTCTGGACCTGGCTAAGGAACTGCTGAACATGTTCACCGCTAACAAAATGGCTAAGGTTTTCTTCACCAACTCTGGTTCTGAAGCTAACGACACCCAGGTTAAAATGGTTTGGTACTACAACAACGCTCTGGGTCGTCCGCAGAAAAAAAAAATCATCGCTCGTGCTAAAGCTTACCACGGTTCTACCTACATCTCTGCTGGTCTGTCTGGTCTGCCGCCAATGCACCAGAAATTCGATTTGCCGCCTCCATTCGTCCTGCACACCGAATGCCCGCACTACTGGGCTTACCACCTGCCGGGTGAGACCGAGGAAGAGTTCTCTACCCGTCTGGCTAATAACCTGGAATCTCTGATCCTGAAAGAAGGTCCGGAAACCGTTGCGGCTTTCATCGCTGAACCGGTTCTGGGTGCTGCTGGTGTTATCCTTCCGCCGGCTACCTACTTCGACAAAGTTCAGGCTATCCTGCGTAAACACGACATCCTGTTCATCGCTGACGAAGTTGTTTGCGGTTTCGGTCGTCTGGGTACCATGTTCGGTTCTGACAAATACAACATCAAACCGGACCTGGTTTCTGTTGGTAAAGCTCTGTCTTCTGGTTACATGCCGATCGCTGCTGTTCTGGTTTCTCAGAAGATCTCTTCTGTTATCCTGTCTGAATCTAACAAAATCGGTGCTTTCTGCCACGGTTTCACCTACTCTGGTCACCCGGTTGCTTGCGCTGTTGCTCTGGAAGCTCTGAAGATTTACAAAGAACGTAACATCACTGAAGTTGTTAACAAGATCTCTCAGAAATTCCAGGAAGGTCTGAAAGCTTTCGCTGACTCTCCGATCATCGGTGAAATCCGTGGTACCGGTCTGGCTCTGTCTACCGAATTCGTTGACAACAAATCTCCGAACGACCCGTTTCCGTACGAATGGGCTGTTGGTACCTACTTCGGTGCTCAGTGCGCTAAATACGGTATGCTGGTTTCTTCTACCGGTGACCACGTTAACATGGCTCCGCCGTTCATCCTGTCTCTGGAAGAACTGGACGAACTGATCCGTATCTACGGTAAAGCTCTGAAAGACACCGAGAAACGTGTTGAAGAACTGAAATCTCAGAAGCAGTGA C. violaceum CV2025 AAQ59697.1 ATGCAAAAACAAAGGACAACTTCACAGTGGCGCGAACTGGATGCAGCGCACCACCTGCATCCGTTCACTGATACCGCGTCGTTGAATCAGGCGGGTGCTCGCGTCATGACCCGTGGCGAGGGCGTCTATCTGTGGGACAGCGAAGGTAATAAGATCATCGATGGTATGGCCGGTTTATGGTGCGTTAACGTGGGTTATGGTCGTAAAGACTTCGCCGAAGCGGCACGCAGACAAATGGAAGAACTTCCGTTTTATAACACCTTTTTTAAAACCACCCATCCGGCGGTGGTGGAATTGAGCTCCCTGTTAGCTGAGGTTACCCCGGCGGGCTTCGATCGTGTTTTTTACACCAACAGCGGTTCCGAGTCCGTGGATACCATGATTCGTATGGTACGCCGTTATTGGGATGTCCAAGGTAAGCCGGAAAAGAAAACTCTTATCGGTCGTTGGAATGGTTACCATGGCAGCACCATTGGTGGCGCGTCTCTGGGTGGTATGAAATACATGCATGAACAGGGCGACCTGCCGATTCCGGGTATGGCTCACATTGAACAACCGTGGTGGTATAAACACGGTAAGGACATGACGCCGGACGAGTTTGGTGTAGTTGCTGCGCGTTGGCTGGAAGAGA81 AAATCCTGGAGATCGGCGCGGATAAAGTTGCGGCGTTCGTGGGCGAGCCGATTCAGGGTGCGGGTGGCGTCATTGTGCCGCCAGCTACCTACTGGCCTGAAATCGAACGTATTTGCCGTAAGTACGATGTGCTGCTGGTGGCGGACGAGGTGATCTGCGGCTTCGGCAGAACAGGTGAGTGGTTTGGCCACCAGCATTTTGGCTTCCAGCCGGACCTGTTTACGGCAGCCAAGGGTTTAAGCAGCGGCTACCTGCCGATCGGCGCTGTGTTCGTTGGCAAGCGTGTTGCTGAGGGCTTGATCGCAGGCGGTGATTTCAACCACGGCTTCACGTACAGCGGTCACCCGGTTTGTGCGGCGGTCGCCCACGCAAATGTAGCGGCGCTCCGTGACGAGGGTATTGTTCAACGTGTTAAAGATGATATTGGACCGTATATGCAGAAACGCTGGCGTGAAACCTTTTCTCGTTTTGAACATGTTGACGACGTGCGTGGTGTGGGTATGGTTCAGGCATTCACGTTGGTTAAGAACAAGGCGAAGCGCGAGTTGTTTCCGGATTTCGGTGAAATCGGCACCCTGTGTCGTGACATCTTCTTCCGCAACAACCTGATCATGCGTGCCTGCGGTGACCACATTGTGAGCGCACCACCGCTGGTCATGACCCGTGCAGAGGTTGACGAGATGCTGGCGGTTGCCGAGCGCTGCTTGGAAGAGTTCGAGCAAACCCTGAAAGCTCGCGGTCTGGCCTAA SUMO Tag AAB01675.1 ATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATACAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGT C. chinense AT3 AAV66309.1 ATGGCTTTCGCTCTGCCGTCTTCTCTGGTTTCTGTTTGCGACAAATCTTTCATCAAACCGTCTTCTCTGACCCCGTCTAAACTGCGTTTCCATAAGCTGAGCTTCATCGACCAGTCGCTGTCTAACATGTACATCCCGTGCGCTTTCTTCTACCCGAAAGTTCAGCAGCGTCTGGAAGACTCTAAAAACTCTGACGAACTGTCTCACATCGCTCACCTGCTGCAGACCTCTCTGTCTCAGACCCTGGTTTCTTACTACCCGTACGCTGGTAAACTGAAAGACAACGCTACCGTTGACTGCAACGACATGGGTGCTGAATTCCTGTCTGTTCGTATCAAATGCTCTATGTCTGAAATCCTGGACCACCCGCACGCTTCGTTAGCGGAATCTATCGTTCTGCCTAAGGACCTGCCGTGGGCCAATAACTGCGAAGGTGGTAACCTGCTGGTTGTTCAGGTTTCTAAATTCGACTGCGGTGGTATCGCTATCTCTGTTTGCTTCTCTCACAAAATTGGTGACGGTTGCTCTCTGCTGAACTTCCTGAACGACTGGTCTTCTGTTACCCGTGACCACACCACCACCGCTCTGGTTCCGTCTCCGCGTTTCGTTGGCGACTCTGTGTTCAGCACCAAAAAGTACGGTTCTCTGATCACCCCGCAGATCCTGTCTGACCTGAACGAATGCGTTCAGAAACGTCTGATCTTCCCGACCGACAAACTGGACGCTCTGCGTGCTAAAGTTGCTGAGGAATCTGGTGTTAAAAACCCGACCCGTGCTGAAGTTGTTTCTGCTCTGCTGTTCAAATGCGCTACCAAAGCTTCTTCTTCTATGCTGCCGTCTAAACTGGTTCACTTCCTGAACATCCGTACCATGATCAAACCGCGTCTGCCGCGTAACGCTATCGGTAACCTGTCTTCTATCTTCTCTATAGAAGCTACCAACATGCAGGACATGGAACTGCCGACCCTGGTTCGTAACCTGCGTAAAGAAGTTGAAGTTGCTTACAAAAAAGACCAGGTTGAACAGAACGAACTGATCCTGGAAGTTGTTGAATCTATGCGTGAGGGTAAACTGCCGTTCGAAAACATGGACGGTTACGAAAACGTTTACACCTGCTCTAACCTGTGCAAATATCCGTACTACACCGTTGACTTCGGTTGGGGTCGTCCGGAACGTGTTTGCCTGGGTAACGGTCCGTCTAAAAACGCTTTCTTCCTGAAAGACTACAAAGCCGGTCAGGGTGTTGAAGCTCGTGTTATGCTGCACAAGCAGCAGATGTCTGAATTCGAACGTAACGAAGAACTGCTGGAATTCATCGCTTAA 82 Supplementary Table 6. Fatty Acid Yield Quantification SF211 - 1 mM IPTG Elution Time Peak Area Normalized Peak Area Against Heptanoic Acid (C7:0) C6:0 6.31 149126144 5.407301238 C7:0 7.66 27578664 1 C8:0 9.33 560037760 20.30692132 C10:0 11.72 67006660 2.429655766 C10:1 11.78 14446022 0.523811523 C12:1 13.92 72011616 2.611135043 C12:0 14.08 17442368 0.632458773 C14:1 16.12 3565331 0.129278597 C14:0 16.28 2396805 0.086907944 Sum - 1 = 32.12 32.12 x 3.9 mg/L = 125 mg/L SF211 - 0 mM IPTG Elution Time Peak Area Normalized Peak Area Against Heptanoic Acid (C7:0) C6:0 6.12 6958300 0.192057943 C7:0 7.62 36230212 1 C8:0 9.02 55403084 1.529195689 BF211 - 1 mM IPTG Elution Time Peak Area Normalized Peak Area Against Heptanoic Acid (C7:0) C5:0 ante-iso 5.12 242927040 6.717870555 C6:0 iso 5.8 3738078 0.103372289 C6:0 ante-iso 5.93 752496 0.02080942 C6:0 6.23 11962450 0.330807927 C7:0 ante-iso 7.32 24569636 0.679445295 C7:0 7.68 36161316 1 C8:0 ante-iso 8.53 5701456 0.157667271 C8:0 9.09 39751856 1.099292293 C9:0 ante-iso 9.96 17953848 0.496493214 C9:0 10.06 18275522 0.505388742 C10:0 ante-iso 11.26 10104596 0.279431092 C10:0 11.69 2316150 0.06405049 C11:0 ante-iso 12.47 3917838 0.108343347 C11:0 iso 12.59 27638520 0.764311786 C12:1 13.92 3413575 0.094398528 C12:0 14.11 1561092 0.043170221 C14:1 16.15 1009609 0.027919587 C14:0 16.31 1871217 0.051746374 C18:0 16.97 582918 0.016119933 Sum - 1 = 11.56063836 11.56 x 3.9 mg/L = 45 mg/L BF211 - 0 mM IPTG Elution Time Peak Area Normalized Peak Area Against Heptanoic Acid (C7:0) C6:0 6.18 4101027 0.185535099 C7:0 7.66 22103780 1 C8:0 9.05 26213668 1.18593598 83 Supplementary Figure 1. GC-MS Chromatograms SF211 - 1 mM IPTG SF211 - 0 mM IPTG b) a) 84 BF211 - 1 mM IPTG BF211 - 0 mM IPTG 2-methylbutanoic acid eluting immediately after the solvent front c) d)
Thesis/Dissertation
eng
Genome Science and Technology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Yadav, Vikramaditya G.
Evaluating E. coli as a chassis for the production of capsaicinoids and capsinoids
Text