Joerg Bohlmann


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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Exploring Mycobacterium tuberculosis Pks12 as a starting point for modular polyketide synthase platform development (2022)

Polyketides are a natural chemical class of complex, frequently bioactive molecules, from which several therapeutics have been derived. Large enzyme complexes, polyketide synthases, are responsible for constructing the carbon backbone of polyketides from simple metabolic precursors. For modular polyketide synthases (mPKSs), the chemical tailoring of each precursor incorporated is controlled by a distinct cluster of domains. These modules are organized as an assembly line. Because the chemistry of the final polyketide chain reflects the organization of modules on the assembly line, mPKSs have become targets for rational protein engineering. If module types could be functionally rearranged in any order, this would expand control over polyketide biosynthetic pathways, enabling access to libraries of novel polyketides from which new antibiotics or therapeutics may be derived. However, combinatorial use of individual mPKS modules has not surpassed 3-module assembly lines. Interactions between the acyl carrier protein (ACP) and ketosynthase (KS) module domains have been particularly sensitive to mismatched interfaces, and these largely constrain use of engineered modules to their specific locations in their natural mPKS assembly lines. ACP-KS interactions remain challenging to model and engineer. In this thesis, I propose an alternative mPKS platform using modules from Mycobacterium tuberculosis PKS12. This bimodular PKS uses repeating, identical KS-ACP interfaces to form 10-module multimers which build long, saturated carbon chains. This makes PKS12 modules attractive templates for creating more interchangeable modules. A prerequisite to developing a PKS12-derived combinatorial platform is the affixing of chain-onloading and chain-offloading domains onto PKS12 modules, enabling it to produce fatty acids. I show that a fatty-acid-producing mPKS can be created from PKS12 parts, and identified an unexpected fusion point for the offloading domain. I identified a plasmid system and culture conditions enabling detectable and reproducible output from PKS12-derived assembly lines in E. coli. I then used this system to demonstrate that antiparallel SYNZIP domains can functionally reattach saturating module halves, providing a new principle for combinatorial mPKS engineering. Finally, I investigate the structural basis for the unexpected C-terminal fusion point, and identify a particular ACP region, helix 2, as a likely contributor.

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Genomics of western redcedar (Thuja plicata) (2022)

Western redcedar (WRC; Thuja plicata) is an ecologically, culturally, and economically important tree species. It is a key successional tree species and a major component of old-growth stands in its range, has been revered and used by First Nations of the Pacific Northwest of North America for millennia, and represents a $1.1B industry. WRC is unique among conifers in that it readily self-fertilizes (selfs) and does not appear to be affected by inbreeding depression for most fitness traits. Further, its tolerance of environmental stressors makes it a focal tree species for future range expansion due to climate change. Development of genomic resources and methods for conifers and other trees has become fundamental over the last decade for better understanding their biology and expediting tree improvement programs for growth and defense traits. As operational forestry shifts from old-growth to second-growth harvesting, using genomic data to better understand the population genetics, demography, and important growth and defense traits of WRC is essential. In this thesis I present novel genomic resources for WRC. These are then used to explore genetic diversity, mating system, and terpene chemistry traits associated with defense against ungulate browsing and fungal pathogens. I found that the genetic diversity of WRC is exceptionally low for a continuous tree species of its range size, likely due to range expansion from a single glacial refugium and its unique self-compatibility. Analysis of selfing lines revealed that WRC’s adaptability and responsiveness to selection may be due to balancing selection as a result of its demography. Further, not only does WRC show limited inbreeding depression for growth, terpene chemistry, or dendrochronological traits, but appropriate selection intensity can mitigate the effects of inbreeding for growth traits. I found that terpene synthase (TPS) genes in WRC occupy a unique clade from other conifers, and that many loci were associated with terpene chemistry traits in WRC, with substantial variation in effect sizes of loci between different traits. This work serves as a foundation for future genomic research, and provides fundamental data for improving breeding prospects for traits of interest in WRC.

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Terpene and isoprenoid biosynthesis in Cannabis sativa (2020)

Cannabis sativa (cannabis, marijuana, hemp) is a plant species grown widely for its psychoactive and medicinal properties. Cannabis products were made illegal in most of the world in the early 1900s, but regulations have recently been relaxed or lifted in some jurisdictions, notably Canada and parts of the United States. Cannabis is usually grown for the resin produced in trichomes on the flowers of female plants. The major components of that resin are isoprenoids: cannabinoids, monoterpenes, and sesquiterpenes. Terpene profiles in cannabis flowers can vary widely between cultivars. My research addresses the genomic underpinnings and biochemical mechanisms of terpene and cannabinoid biosynthesis in cannabis, and patterns of terpene accumulation between organs, developmental stages, and cultivars. Using metabolite profiling, I demonstrated that terpenes accumulate in floral trichomes over the course of development, and that terpene profiles in trichomes differ based on tissue and developmental stage. In this thesis, I describe the terpene profiles of seven cannabis cultivars. I identified and characterized 29 terpene synthase (TPS) genes and their encoded enzymes and describe the relationship between TPS expression and metabolite profiles. I describe trichome-specific transcriptomes for five cultivars and identify highly expressed genes common to cannabis trichomes. I also identified and describe an aromatic prenyltransferase responsible for biosynthesis of cannabigerolic acid, the branch-point intermediate in cannabinoid biosynthesis. Collectively, this thesis comprises a broad and detailed characterization of specialized isoprenoid biosynthesis in cannabis. The results provide new insights into mechanisms of terpene and cannabinoid biosynthesis, and the roles of different enzymes in determining the metabolite complement of cannabis trichomes.

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Detoxification of pine terpenoids by mountain pine beetle cytochromes P450 (2019)

The mountain pine beetle (Dendroctonus ponderosae; MPB) is an irruptive bark beetle species affecting pine forests of western North America. A recent outbreak has spread over more than 25 million hectares of pine forests now affecting pine species of sensitive boreal and mountain ecosystems. Pine hosts produce a viscous oleoresin comprised of terpenoids; monoterpene, sesquiterpenes and diterpene resin acids, as a defense against insects and other herbivores. The MPB is exposed to terpenoids for most of its life cycle and these compounds act as host defenses, kairomones, and as pheromone precursors. Cytochromes P450 (P450s) have been proposed to function in MPB detoxification of host defenses, olfaction, and pheromone biosynthesis. My research addressed the role of terpenoids as toxic host defenses and pheromone precursors, and the role of MPB P450s in the modification of terpenoids in detoxification, odorant degradation, and pheromone biosynthesis.In this thesis, I assessed the toxic effects of monoterpenes to MPB, and analyzed the terpenoid metabolic products formed by MPB and by MPB P450s from host monoterpenes and diterpene resin acids. I assessed the toxicity of ten monoterpenes to the MPB. This study helps to quantitatively define the effects of individual monoterpenes towards MPB mortality, which is critical when assessing the variable monoterpene chemical defense profiles of its host species. I identified a set of novel monoterpenyl esters in the MPB, including verbenyl ester and showed that these esters are accumulated by the female beetle early in the life cycle for future release of the MPB aggregation pheromone trans-verbenol. I investigated seven different MPB P450s, specifically CYP6DE1, CYP6DE2, CYP6DJ1, CYP6BW1, CYP6BW3, CYP9Z18 and CYP345E2, for their potential roles in detoxification, odorant degradation, and pheromone biosynthesis by quantifying the transcript abundance in the antennae and alimentary canal. I have characterized the biochemical functions of four of these MPB P450s. The results of my thesis provide new insights into MPB interactions with host terpenoids defenses and the roles of P450s in these interactions.

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Efficient assembly of large genomes (2019)

Genome sequence assembly presents a fascinating and frequently-changing challenge. As DNA sequencing technologies evolve, the bioinformatics methods used to assemble sequencing data must evolve along with it. Sequencing technology has evolved from slab gel sequencing, to capillary sequencing, to short read sequencing by synthesis, to long-read and linked-read single-molecule sequencing. Each evolutionary jump in sequencing technology required developing new bioinformatic tools to address the unique characteristics of its sequencing data. This work reports the development of efficient methods to assemble short-read and linked-read sequencing data, named ABySS 2.0 and Tigmint. ABySS 2.0 reduces the memory requirements of short-read genome sequencing assembly by ten fold compared to ABySS 1.0. It does so by using a Bloom filter probabilistic data structure to represent a de Bruijn graph. Tigmint uses linked reads to identify large-scale errors in a genome sequence assembly. Correcting assembly errors using Tigmint before scaffolding improves both the contiguity and correctness of a human genome assembly compared to scaffolding without correction. I have also applied these methods to assemble the 12 gigabase genome of western redcedar (Thuja plicata), which is four times the size of the human genome.Although numerous mitochondrial genomes of angiosperm are available, few mitochondria of gymnosperms have been sequenced. I assembled the plastid and mitochondrial genomes of white spruce (Picea glauca) using whole genome short read sequencing. I assembled the mitochondrial genome of Sitka spruce (Picea sitchensis) using whole genome long read sequencing, the largest complete genome assembly of a gymnosperm mitochondrion. The mitochondrial genomes of both species include a remarkable number of trans-spliced genes.I have developed two additional tools, UniqTag and ORCA. UniqTag assigns unique and stable gene identifiers to genes based on their sequence content. This gene labeling system addresses the inconvenience of gene identifiers changing between versions of a genome assembly. ORCA is a comprehensive bioinformatics computing environment, which includes hundreds of bioinformatics tools in a single easily-installed Docker image, and is useful for education and research.The assembly of linked read and long read sequencing of large molecules of DNA have yielded substantial improvements in the quality of genome assembly projects.

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Elucidation and characterization of genes associated with montbretin a biosynthesis within crocosmia x crocosmiiflora and white pine weevil defense in picea sitchensis (2018)

Plant-specialized metabolites have long been utilized as medicines, cosmetics, flavours, and industrial raw materials. To explore the biosynthesis of a specialized metabolite in a non-model system and utilize the biosynthetic genes for future application, genomics-informed research typically flows through three phases: i) development of genomic or transcriptomic resources, ii) discovery and characterization of biosynthetic genes, and iii) application of the genes and enzymes for improved production of the specialized metabolite. This thesis describes hypothesis-driven research along these three phases in two different plant species and two different metabolic systems. My research with Crocosmia x crocosmiiflora focused on resource development and discovery of biosynthetic genes of a specialized metabolite of interest, montbretin A (MbA). I developed new resources for this system including metabolite-profiles and transcriptome sequences and annotations. This work resulted in insight into the spatial and temporal patterns of MbA accumulation in C. x crocosmiiflora and a first reference transcriptome with annotation for this species. Using these resources, I functionally characterized four UDP-xylose synthases and five UDP-rhamnose synthases. I discuss the application of these genes for possible use in an improved MbA production system and provide a proof of concept for using these genes to enable characterization of downstream MbA biosynthetic genes. I also identified 14 UDP-glycosyltransferases as candidate MbA biosynthetic genes through a guilt-by-association analysis; however, their functional characterization did not support a role in MbA biosynthesis. In the second biological system, Sitka spruce (Picea sitchensis), I performed a detailed characterization of a set of monoterpene synthases involved in the biosynthesis of the (+)-3-carene. Using domain swapping and site-directed mutagenesis, I demonstrated the catalytic plasticity of monoterpene synthases across a family of (+)-3-carene synthase-like genes associated with P. sitchensis resistance against the white pine weevil (Pissodes strobi). This work identified a single amino acid as most critical in determining both product profile and enzyme kinetics. Furthermore, I described mechanisms by which this amino acid directs product profiles through differential stabilization of the reaction intermediate. The work presented highlights the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against pests.

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Functional Characterization and Spatial and Temporal Patterns of Expression of Genes Involved in Gibberellin and Diterpene Resin Acid Biosynthesis in White Spruce (2016)

Conifers produce large quantities of diterpene resin acids (DRAs) as major components of the constitutive and induced oleoresin defense system. Like all vascular plants, conifers also produce gibberellin (GA) diterpene phytohormones, which influence growth and development. Conifers thus provide an interesting biological system for comparing the GA and DRA diterpene biosynthetic pathways. Despite serving different functions in growth and defense, respectively, the GA and DRA biosynthetic pathways are biochemically similar, utilizing the same isoprenoid precursors, evolutionarily related diterpene synthases (diTPSs), and functionally similar cytochrome P450 monooxygenases (CYP450s) to produce structurally similar diterpene intermediates and diterpene acid products. Functional characterization of central diTPS genes (ent-copalyl diphosphate synthase [CPS], ent-kaurene synthase [KS], levopimaradiene/abietadiene synthase [LAS]) and CYP450 genes (ent-kaurene oxidase [CYP701] and CYP720B4) in white spruce (Picea glauca), described in this thesis, allowed for comparative analysis of GA and DRA pathway genes. This thesis characterized the DXS (1-deoxy-D-xylulose 5-phosphate synthase) gene family in white spruce as additional analysis of the isoprenoid biosynthetic pathway producing the common precursor to both GAs and DRAs. Transcript expression of genes was analyzed to understand their seasonal (year-long time course of apical bud and shoot development), sample-specific (e.g. needle, stem, bud, bark/phloem, wood/xylem), and stress-specific (methyl jasmonate [MeJA] exposure) spatial and temporal patterns. Functionality of the DRA pathway was also assessed via quantification of DRA products. Expression of DRA and GA pathway genes was generally spatially separated. Expression of DRA genes was low in photosynthetic tissues but up-regulated during the time of year when trees are most likely to encounter seasonal attack from insect pests; expression declined sharply well before dormancy showing a strong seasonality to DRA production. GA related genes had broader expression across sample types and throughout the year, but spatially were mainly allocated to photosynthetic tissues. GA and DRA pathway genes all showed differential responses to MeJA treatment, and within corresponding sample types, age also played a role in expression. These studies improve our understanding of the organization of conifer chemical defenses, showing distinct differences compared with GA gene expression, and providing information on the spatial, seasonal and stress-responsive expression of DRA pathway genes.

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Transcriptome analysis of conifer defense against bark beetle-associated blue-stain fungi and white pine weevil (2011)

Conifer forests are exposed to a large number of herbivorous insect species and pathogenic fungi, some of which cause extensive epidemics and substantial losses of forest resources. Bark beetles and white pine weevil represent major threats to conifer forest health. Bark beetles vector fungal pathogens, which are involved in killing of the host trees. Conifers employ a variety of defense strategies, including anatomical, chemical and molecular defense mechanisms. Recent development of conifer genomic resources and tools including large EST databases and microarrays have allowed for large-scale analysis of conifer defense. To evaluate transcriptome response of conifer species to fungal pathogens I performed a comparative analysis of the interior spruce (Picea glauca x engelmannii) response to spruce beetle-associated pathogenic blue-stain fungus Leptographium abietinum and the lodgepole pine (Pinus contorta) response to mountain pine beetle-associated pathogenic blue-stain fungus Grosmannia clavigera using a 21,843-clone cDNA spruce microarray platform. In addition, I performed a direct comparison of the interior spruce response to inoculation with the fungus Leptographium abietinum with the response to white pine weevil (Pissodes strobi) herbivory. The microarray analyses revealed substantial changes in the transcriptomes of conifer hosts in response to fungal inoculation or insect feeding with more than a thousand genes significantly differentially expressed in each system and interaction studied. The fungus-induced transcriptomes of spruce and pine shared a large number of similarly responding transcripts with some differences in the dynamics of the induced responses. The transcriptome responses of spruce induced by fungal inoculation and weevil feeding had a large overlap and some treatment-specific trends. Among the most strongly up-regulated transcripts in all interactions were phenylpropanoid pathway transcripts, dirigent protein transcripts, laccases, chitinases and transcripts of the terpenoid pathway. Gene specific expression analysis of selected transcripts confirmed and extended the microarray analysis. Cloning and functional characterization of selected chitinases revealed the presence of chitinolytic activity in two interior spruce and one lodgepole pine class I chitinases. Chitinolytic activity in addition to the strong induction of these chitinases in response to different treatments supported their involvement in conifer defense.

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Terpenoid profiling and biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are susceptible or resistant to attack by the white pine weevil (Pissodes strobi) (2010)

White pine weevil, Pissodes strobi, is an insect that occurs throughout Canada that attacks a number of conifers including Sitka spruce, Picea sitchensis, a commercially and ecologically important tree for coastal B.C.. Because of attack by weevils, Sitka spruce is no longer replanted as a commercial species. The re-introduction of this species would be a valuable asset for sustainable coastal forestry. My research addresses the terpene composition and the molecular-genetic underpinning of Sitka spruce resin defenses against attack by white pine weevil. In this thesis, I report that terpene profiles can be used to classify resistant tree genotypes. I analysed 111 different genotypes in order to determine the relationship of mono- and diterpenoid oleoresin compounds with the resistance rating. Dehydroabietic acid, a diterpene, was identified as a strong indicator of resistance. Two monoterpenes, (+)-3-carene and terpinolene were also associated with resistance in genotypes originating from the Haney region, an area which may have been subject to higher weevil pressure. In addition, I characterized weevil behavior and physiology (feeding patterns, host choice, ovary development, egg laying behavior, and larval development) in response to an extremely resistant Sitka spruce genotype (H898) in comparison to a highly susceptible genotype (Q903). My results suggest that the highly resistant genotype H898 has defense mechanisms that deter both male and female weevils during host selection and mating, that cause delayed ovary development in females, and prevent successful reproduction of weevils on H898 trees. Finally, I have identified the first (+)-3-carene and (+)-sabinene synthase genes in Sitka spruce. These terpene synthase (TPS) genes have very similar sequences, yet the encoded enzymes have different product profiles; this shows a new level of genetic diversity in the spruce TPS gene family. In addition, different (+)-3-carene synthase genes are expressed in the resistant H898 tree genotype producing large amounts of (+)-3-carene, versus the susceptible Q903 tree genotype that produces trace amounts of (+)-3-carene. This information will support the identification and breeding of resistant Sitka spruce in order to re-introduce it as a viable, native commercial species.

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Genomics of systemic induced defense responses to insect herbivory in hybrid poplar (2008)

The availability of a poplar (Populus trichocarpa Torr & A. Gray, black cottonwood) genomesequence is enabling new research approaches in angiosperm tree biology. Much of the recentgenomics research in poplars has been on wood formation, growth and development, and abiotic stresstolerance, motivated, at least in part, by the fact that poplars provide an important system for largescale, short-rotation plantation forestry in the Northern Hemisphere. Given their widespreaddistribution and long lifespan, poplar trees are threatened by a large variety of insect herbivore pests,and must deal with their attacks with a successful defense response. To sustain productivity andecosystem health of natural and planted poplar forests, it is of critical importance to develop a betterunderstanding of the molecular mechanisms of defense and resistance of poplars against insect pests.Previous research has established a solid foundation of the chemical ecology of poplar defense againstinsects. In this study, I buiLd on this base with large-scale profiling of transcriptome responses ofpoplar trees to insect herbivory. A 15,496-clone cDNA microarray was developed and used to analysetranscriptome responses through time to a variety of insect, mechanical, and chemical elicitortreatments in treated source leaves, as well as in undamaged systemic source and sink leaves of hybridpoplar (Populus trichocarpa x deltoides).Comparing mechanical wounding with insect feeding and chemical eLicitor treatment withmethyl jasmonate demonstrated that qualitatively similar profiles of transcriptome response wereeLicited with differences in the timing of induction. Transcriptome analysis in undamaged systemicleaves of treated trees uncovered distinct early changes in primary metabolism (e.g. sugar metabolism)and general stress responses (e.g. heat shock proteins) prior to the activation of insect herbivoryresponse genes (e.g. Kunitz-type protease inhibitors). Source-sink relationships are maintained andstrengthened by insect damage on source leaves, emphasizing changes in resource allocation patternsas being important for poplar defense. Overall, a model of poplar defense begins to emerge where acascade of transcriptome profiles through space and time lead to reorganization of metabolism fortolerance and induction of defense.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Engineering flavanone biosynthesis in yeast towards the production of montbretin A (2022)

Plants produce a diverse range of specialized metabolites, which are an important source for new pharmaceuticals. The plant metabolite montbretin A (MbA) is a novel therapeutic for type 2 diabetes. MbA is a complex acylated flavonol glycoside found in the underground storage organs, called corms, of the ornamental plant montbretia (Crocosmia x crocosmiiflora). Large quantities of MbA are needed to treat the millions of people who would benefit from it. However, MbA cannot be extracted from montbretia corms in sufficient quantities for therapeutic use, and chemical synthesis of MbA is not viable due to the compound’s chemical complexity. Engineering a plant or microbial system for large-scale, sustainable MbA production would be a viable option. The recent elucidation of the complete MbA biosynthetic pathway provides the tools to engineer yeast (Saccharomyces cerevisiae) for heterologous MbA production. The core of MbA is the flavonol myricetin, the biosynthesis of which needs to be bioengineered in yeast. In most plants, biosynthesis of myricetin uses coumaric acid as a substrate and produces naringenin as a flavanone intermediate. However, utilizing metabolic engineering of myricetin from coumaric acid via naringenin poses a problem for the downstream bioengineering of MbA in yeast. The presence of coumaric acid would lead to formation of MbB instead of MbA, where MbB contains a coumaroyl side chain instead of the caffeoyl side chain in MbA. MbB is an undesired by-product with no known therapeutic value. To address this problem, I developed a yeast strain that uses caffeic acid (instead of coumaric acid) in the formation of myricetin via eriodictyol (instead of naringenin). I achieved the formation of eriodictyol as a precursor to myricetin at 27.3 mg * L⁻¹. Optimizing flavonoid enzyme combinations, tuning gene expression, and the addition of the chalcone isomerase-like (CHIL) protein improved eriodictyol production up to 8-fold. Overall, this work demonstrates the potential of engineering a yeast strain to produce the flavanone eriodictyol, an essential step towards scalable and sustainable production of the anti-diabetic plant metabolite MbA.

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Engineering montbretin A biosynthesis in Nicotiana benthamiana (2022)

The plant kingdom has always been a useful source of therapeutic molecules for humans. Montbretin A (MbA) is a complex acylated flavonol glycoside found in the underground storage organs, called corms, of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA was discovered as a highly efficient and specific inhibitor of the human pancreatic α-amylase (HPA), making it an attractive candidate for the treatment of type 2 diabetes, a worldwide epidemic. The original plant source montbretia is not adapted for large scale cultivation, and MbA’s complex structure renders chemical synthesis infeasible for scalable production. Therefore, we are aiming to engineer a heterologous system for large-scale sustainable MbA production for treatment of type 2 diabetes. To this end we are using synthetic biology to develop an MbA producing Nicotiana benthamiana (tobacco) strain. This is possible due to the recent complete elucidation of the MbA biosynthetic pathway, including all essential genes and enzymes from montbretia.Small amounts of MbA can be produced in N. benthamiana through transient expression of the MbA biosynthetic enzymes. However, much higher quantities of montbretin B (MbB) and montbretin C (MbC), which differ from MbA in the identity of their acyl groups, were produced. MbB and MbC possess a coumaroyl and a feruloyl group, respectively, and are not effective HPA inhibitors. To influence acyl group availability, specifically increase the amount of caffeoyl-CoA for MbA biosynthesis, we investigated a set of enzymes involved in caffeoyl-CoA biosynthesis in montbretia, specifically 4-coumaroyl CoA ligases (4CL), hydroxycinnamoyl- CoA shikimate/quinate hydroxycinnamoyltransferases (HCT), 4-coumarate 3-hydroxylases (C3Hs), and caffeoyl-shikimate esterases (CSE). Co-expression of those caffeoyl-CoA biosynthetic genes together with MbA biosynthetic genes resulted in up to 10-fold higher MbA yields.

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Thujone biosynthesis in western redcedar (Thuja plicata) (2020)

Western redcedar (WRC; Thuja plicata) is a highly valued conifer species native to the west coast of North America. However, herbivore feeding, specifically browsing by deer and elk, is a major problem for WRC reforestation. Browsing deterrence correlates with high levels of foliar monoterpenes, particularly α- and β-thujone, which accumulate in resin glands in WRC foliage and seeds. Thujone is proposed to derive from geranyl diphosphate via a four-step biosynthetic pathway. The first two enzymes of the pathway have recently been identified: a monoterpene synthase converts geranyl diphosphate into (+)-sabinene, which is oxidized into (+)-trans-sabinol through a cytochrome P450. It is hypothesized that (+)-trans-sabinol is further oxidized to (+)-sabinone and then reduced to α- and β-thujone.In this study, I further investigated thujone biosynthesis in WRC. First, I monitored thujone pathway intermediates and resin storage structures throughout the growing season. Both foliage and seed cones had a temporal shift from sabinene to α-thujone as the predominant monoterpene. This shift in monoterpene predominance was accompanied by the deterioration of epithelial cells surrounding oleoresin storage structures. Second, I selected candidate genes for the third step of the thujone pathway, proposed to be an alcohol dehydrogenase (ADH) catalyzing the oxidation of (+)-trans-sabinol to (+)-sabinone. Using transcriptomic analysis of wild type WRC foliage as well as a naturally occurring WRC variant that does not produce monoterpenes, I identified 21 putative ADHs. Third, I tested candidate ADH gene functions through in vitro assays with recombinant protein that was heterologously expressed in E. coli. Three ADHs (ADH5, ADH10, and ADH17) were found to have activity, producing β-thujone rather than the expected product of (+)-sabinone. Optimization in protein expression and activity assay conditions led to ADH5 producing both β-thujone and (+)-sabinone as major products.Understanding the genes involved in thujone biosynthesis will provide valuable information for WRC tree breeding. Discovering mechanisms of resistance in WRC will support efforts to produce seedlings that are more resistant to ungulate browsing and have the desired resistance attributes for reforestation.

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Investigating montbretin A biosynthesis and elucidating acyltransferase in Crocosmia x crocosmiiflora (2018)

Plant specialized metabolites have been historically used in traditional medicine, flavours and fragrances throughout centuries, and they still serve as a valuable source for new pharmaceutical and nutraceutical development. Montbretin A (MbA) is an acylated flavonol glycoside produced by the ornamental plant montbretia (Crocosmia x crocosmiiflora), and mainly accumulates in the underground storage organs, called corms. This unique metabolite is a highly specific inhibitor of the human pancreatic α-amylase (HPA), thus making it a promising candidate for drug development against type-2 diabetes. However, a production system for obtaining large quantities of MbA is currently unavailable. Metabolic engineering of MbA in alternative microbial or plant systems may lead to the large-scale production of MbA.The main goal of this thesis was to obtain foundational insight on MbA biosynthesis. I first examined the growth and development of the montbretia plant, and performed detailed metabolite analysis focusing on the corms. The results of the metabolite profiling revealed the spatiotemporal patterns of MbA biosynthesis. This provided a foundational knowledge for the development of the montbretia transcriptome by Irmisch et al. (submitted). Furthermore, based on the activity of corm protein extracts, I identified that a member of the BAHD-AT family is involved in the acylation step of MbA biosynthesis. The candidate BAHD-ATs were identified using the established montbretia transcriptome, and were cloned and heterologously expressed in Escherichia coli for functional characterization. Of the seven candidate ATs tested, two candidates, CcAT1 and CcAT2 catalyzed the formation of mini-MbA, the product of the third step of MbA biosynthesis. Additionally, CcAT1 and CcAT2 were transiently expressed in the leaves of Nicotiana benthamiana, which led to the formation of a surrogate mini-MbA compound. This provided preliminary insight towards the metabolic engineering of MbA in N. benthamiana. Furthermore, qRT-PCR analyses were performed to investigate the transcript abundance patterns of CcAT1 and CcAT2 during montbretia corm development. The transcript profiling of CcAT1 and CcAT2 further supported their in vivo roles of MbA biosynthesis. Overall, the results presented in this thesis provide new knowledge on specialized plant metabolism in a non-model plant species.

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Characterization and heterologous expression of cis-abienol synthase from balsam fir (Abies balsamea) towards high-end fragrance components (2014)

Background The cis-abienol synthase (AbCAS) gene of balsam fir (Abies balsamea) encodes a diterpene synthase (diTPS), which produces a compound, cis-abienol, of value for the fragrance industry. The same compound is also produced in Abies lasiocarpa. cis-Abienol can be used as a starting material for semisynthesis of the commercial substance Ambrox® which is used in the formulation of high end perfumes. AbCAS is a bifuncational diTPS which produces cis-abienol in a two-step process involving two active sites responsible for the class I and class II activities. Class-II activity generates the hydroxylated labda-13-en-8-ol diphosphate (LPP) intermediate, followed by the class-I activity which produces the cis-abienol product.Results We investigated the effects that amino acid residue changes have on catalytic activity of AbCAS and related diTPSs and the product profiles of these protein variants. We identified critical amino acid residues (D348H and S345S) that are responsible for product specificity in AbCAS. In addition, in order to determine if the mutations in AbCAS were also relevant to other diTPSs, this study extended investigation into the AbLAS enzyme, a multiproduct levopimaradiene/abietadiene synthase. The AbLAS:C345S:H346D protein variant showed a new activity and produced the oxygenated diterpene manoyl oxide (100% product profile), where as AbLAS (WT) produces exclusively olefins. To develop a metabolic engineering platform for cis-abienol production we investigated yeast (Saccharomyces cerevisiae) and in the bryophyte Physomitrella patens as hosts for expression of AbCAS. We examined expression of AbCAS with three different S. cerevisiae strains (Am94, BY4741 and KE Strain) and successfully produced cis-abienol in all three strains with up to 12.8 mg x L-¹ in the AM94 strain. Physcomitrella transformed with AbCAS did not produce detectable levels of cis-abienol. Conclusions Product specificity of the diTPS enzymes AbCAS and AbLAS can be altered by changing as few as one or two amino acid residues. Expression of AbCAS in yeast resulted in the formation of cis-abienol indicating opportunities for metabolic engineering of a recombinant production system for this valuable diterpene compound.

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Cutting trees with lasers: isolation of high quality RNA, enzymatically active protein and metabolites from individual tissue types of white spruce stems obtained using laser microdissection (2012)

Laser-assisted microdissection has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in a regular pattern with xylem, phloem and cortex development controlled by the activity of the cambial zone (CZ). Defense responses of conifer stems against insects and pathogens involve increased accumulation of terpenoids in cortical resin ducts (CRDs) and de novo formation of traumatic resin ducts from CZ initials. Woody plants are difficult to study at the level of individual tissues or cell-types and are thus good candidates for application of LMD. This thesis describes robust methods for isolation of individual tissue-types from white spruce (Picea glauca) stems for analysis of RNA, enzyme activity and metabolites. A tangential cryosectioning approach was important for obtaining large quantities of CRD and CZ tissues using LMD. Differential expression is reported for genes involved in terpenoid metabolism between CRD and CZ tissues and in response to treatment with methyl jasmonate (MeJA). Transcript levels of β-pinene synthase and levopimaradiene/abietadiene synthase were constitutively higher in CRDs, but induction was stronger in CZ in response to MeJA. 3-Carene synthase was more strongly induced in CRDs compared to CZ. A differential induction pattern was observed for 1-deoxyxyulose-5-phosphate synthase, which was up-regulated in CRDs and down-regulated in CZ. We identified terpene synthase enzyme activity in CZ protein extracts and terpenoid metabolites in both CRD and CZ tissues. Combined analysis of transcripts, proteins and metabolites of individual tissues will facilitate future characterization of complex processes of woody plant development, including periodic stem growth and dormancy, cell specialization, and defense and may be applied widely to other plant species.

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