Vincent J J Martin

Concordia University Montreal, Montréal, Quebec, Canada

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Publications (48)233.48 Total impact

  • Damien Biot-Pelletier · Vincent J. J. Martin
    No preview · Article · Dec 2016 · Journal of Biological Engineering
  • [Show abstract] [Hide abstract] ABSTRACT: The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anti-cancer drugs vinblastine and vincristine. These compounds are of global significance – appearing on the World Health Organization’s list of model essential medicines – but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. Lastly we demonstrate that activity of the iridoid synthase towards the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the non-productive intermediates citronellol and 10-hydroxycitronellol. This discovery has serious implications for the reconstruction of the MIA in heterologous organisms.
    No preview · Article · Mar 2016 · ACS Synthetic Biology
  • [Show abstract] [Hide abstract] ABSTRACT: Benzylisoquinoline alkaloids (BIAs) constitute a diverse class of plant secondary metabolites that includes the opiate analgesics morphine and codeine. Collectively, BIAs exhibit a myriad of pharmacological activities, including antimicrobial, antitussive, antispasmodic, and anticancer properties. Despite 2500 known BIA products, only a small proportion are currently produced though traditional crop-based manufacturing, as complex stereochemistry renders chemical synthesis of BIAs largely unfeasible. The advent of synthetic biology and sophisticated microbial engineering coupled with recent advances in the elucidation of plant BIA metabolic networks has provided growing motivation for producing high-value BIAs in microbial hosts. Here, we provide a technical basis for reconstituting BIA biosynthetic pathways in the common yeast Saccharomyces cerevisiae. Methodologies outlined in this chapter include fundamental techniques for expressing and assaying BIA biosynthetic enzymes, bioprospecting large libraries of BIA enzyme variants, and reconstituting and optimizing complete BIA formation pathways in yeast. To expedite construction of superior BIA-producing yeast strains, we emphasize high-throughput techniques. Finally, we identify fundamental challenges impeding deployment of yeast-based BIA production platforms and briefly outline future prospects to overcome such barriers.
    No preview · Chapter · Mar 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Benzylisoquinoline alkaloids (BIAs) are a family of ∼2500 alkaloids with both potential and realized pharmaceutical value, including most notably the opiates such as codeine and morphine. Only a few BIAs accumulate readily in plants, which limits the pharmaceutical potential of the family. Shifting BIA production to microbial sources could provide a scalable and flexible source of these compounds in the future. This review details the current status of microbial BIA synthesis and derivatization, including rapid developments in the past 6 months culminating in the synthesis of opioids from glucose in a microbial host.
    No preview · Article · Jan 2016 · Trends in Biotechnology
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    [Show abstract] [Hide abstract] ABSTRACT: L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4 (FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways. Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae.
    Preview · Article · May 2015 · Microbial Cell Factories
  • [Show abstract] [Hide abstract] ABSTRACT: Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.
    No preview · Article · May 2015 · Nature Chemical Biology
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    [Show abstract] [Hide abstract] ABSTRACT: Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.
    Full-text · Article · Apr 2015 · PLoS ONE
  • Damien Biot-Pelletier · Vincent J J Martin
    [Show abstract] [Hide abstract] ABSTRACT: An upsurge in the bioeconomy drives the need for engineering microorganisms with increasingly complex phenotypes. Gains in productivity of industrial microbes depend on the development of improved strains. Classical strain improvement programmes for the generation, screening and isolation of such mutant strains have existed for several decades. An alternative to traditional strain improvement methods, genome shuffling, allows the directed evolution of whole organisms via recursive recombination at the genome level. This review deals chiefly with the technical aspects of genome shuffling. It first presents the diversity of organisms and phenotypes typically evolved using this technology and then reviews available sources of genetic diversity and recombination methodologies. Analysis of the literature reveals that genome shuffling has so far been restricted to microorganisms, both prokaryotes and eukaryotes, with an overepresentation of antibiotics- and biofuel-producing microbes. Mutagenesis is the main source of genetic diversity, with few studies adopting alternative strategies. Recombination is usually done by protoplast fusion or sexual recombination, again with few exceptions. For both diversity and recombination, prospective methods that have not yet been used are also presented. Finally, the potential of genome shuffling for gaining insight into the genetic basis of complex phenotypes is also discussed.
    No preview · Article · Mar 2014 · Applied Microbiology and Biotechnology
  • [Show abstract] [Hide abstract] ABSTRACT: Benzylisoquinoline alkaloids (BIAs) represent a large class of plant secondary metabolites, including pharmaceuticals such as morphine, codeine and their derivatives. Large-scale production of BIA-based pharmaceuticals is limited to extraction and derivatization of alkaloids that accumulate in planta. Synthesis of BIAs in microbial hosts could bypass such limitations and transform both industrial production of BIAs with recognized value and research into uncharacterized BIAs. Here we reconstitute a 10-gene plant pathway in Saccharomyces cerevisiae that allows for the production of dihydrosanguinarine and its oxidized derivative sanguinarine from (R,S)-norlaudanosoline. Synthesis of dihydrosanguinarine also yields the side-products N-methylscoulerine and N-methylcheilanthifoline, the latter of which has not been detected in plants. This work represents the longest reconstituted alkaloid pathway ever assembled in yeast and demonstrates the feasibility of the production of high-value alkaloids in microbial systems.
    No preview · Article · Feb 2014 · Nature Communications
  • [Show abstract] [Hide abstract] ABSTRACT: Use of lignocellulosic biomass as a second generation feedstock in the biofuels industry is a pressing challenge. Among other difficulties in using lignocellulosic biomass, one major challenge is the optimal utilization of both 6-carbon (glucose) and 5-carbon (xylose) sugars by industrial microorganisms. Most industrial microorganisms sequentially utilize glucose over xylose owing to the regulatory phenomenon of carbon catabolite repression (CCR). Microorganisms that can co-utilize glucose and xylose are of considerable interest to the biofuels industry due to their ability to simplify the fermentation processes. However, elimination of CCR in microorganisms is challenging due to the multiple coordinating mechanisms involved. We report a novel algorithm, SIMUP, which finds metabolic engineering strategies to force co-utilization of two sugars, without targeting the regulatory pathways of CCR. Mutants of Escherichia coli based on SIMUP algorithm showed predicted growth phenotypes and co-utilized glucose and xylose; however, consumed the sugars slower than the wild-type. Some solutions identified by the algorithm were based on stoichiometric imbalance and were not obvious from the metabolic network topology. Furthermore, sequencing studies on the genes involved in CCR showed that the mechanism for co-utilization of the sugars could be different from previously known mechanisms.
    No preview · Article · Aug 2013 · Metabolic Engineering
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    [Show abstract] [Hide abstract] ABSTRACT: Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavours, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).
    Full-text · Article · Apr 2013 · Journal of Biotechnology
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    Paramjit K Bajwa · Chi-Yip Ho · Chi-Kin Chan · Vincent J J Martin · Jack T Trevors · Hung Lee
    [Show abstract] [Hide abstract] ABSTRACT: Global gene expression was analyzed in Saccharomyces cerevisiae T2 cells grown in the presence of hardwood spent sulphite liquor (HW SSL) and each of the three main inhibitors in HW SSL, acetic acid, hydroxymethyfurfural (HMF) and furfural, using a S. cerevisiae DNA oligonucleotide microarray. The objective was to compare the gene expression profiles of T2 cells in response to the individual inhibitors against that elicited in response to HW SSL. Acetic acid mainly affected the expression of genes related to the uptake systems of the yeast as well as energy generation and metabolism. Furfural and HMF mainly affected the transcription of genes involved in the redox balance of the cell. On the other hand, the effect of HW SSL on S. cerevisiae T2 cells was distinct and considerably more diverse as compared to the effect of individual inhibitors found in lignocellulosic hydrolysates. This is not surprising as HW SSL contains a complex mixture of inhibitors which may act synergistically. HW SSL elicited significant changes in expression of genes involved in diverse and multiple effects on several aspects of the cellular structure and function. A notable response to HW SSL was decreased expression of the ribosomal protein genes in T2 cells. In addition, HW SSL decreased the expression of genes functioning in the synthesis and transport of proteins as well as metabolism of carbohydrates, lipids, vitamins and vacuolar proteins. Furthermore, the expression of genes involved in multidrug resistance, iron transport and pheromone response was increased, suggesting that T2 cells grown in the presence of HW SSL may have activated pheromone response and/or activated pleiotropic drug response. Some of the largest changes in gene expression were observed in the presence of HW SSL and the affected genes are involved in mating, iron transport, stress response and phospholipid metabolism. A total of 59 out of the 400 genes differentially expressed in the presence of HW SSL, acetic acid, HMF and furfural, belonged to the category of poorly characterized genes. The results indicate that transcriptional responses to individual lignocellulosic inhibitors gave a different picture and may not be representative of how the cells would respond to the presence of all the inhibitors in lignocellulosic hydrolysates such as HW SSL.
    Full-text · Article · Mar 2013 · Antonie van Leeuwenhoek
  • Euan Burton · Vincent J J Martin
    [Show abstract] [Hide abstract] ABSTRACT: Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the ability to directly convert cellulosic biomass into useful products such as ethanol and hydrogen. In this study, a quantitative comparative proteomic analysis of the organism was performed to identify proteins and biochemical pathways that are differentially utilized by the organism after growth on cellobiose or cellulose. The cytoplasmic and membrane proteomes of C. thermocellum grown on cellulose or cellobiose were quantitatively compared using a metabolic (15)N isotope labelling method in conjunction with nanoLC-ESI-MS/MS (liquid chromatography - electrospray ionization - tandem mass spectrometry). In total, 1255 proteins were identified in the study, and 129 of those were able to have their relative abundance per cell compared in at least one cellular compartment in response to the substrate provided. This study reveals that cells grown on cellulose increase their abundance of phosphoenolpyruvate carboxykinase while decreasing the abundance of pyruvate dikinase and oxaloacetate decarboxylase, suggesting that the organism diverts carbon flow into a transhydrogenase-malate pathway that can increase the production of the biosynthetic intermediates NADPH and GTP. Glutamate dehydrogenase was also found to have increased abundance in cellulose-grown cells, suggesting that the assimilation of ammonia is upregulated in cells grown on the cellulosic substrates. The results illustrate a mechanism by which C. thermocellum can divert carbon into alternative pathways for the purpose of producing biosynthetic intermediates necessary to respond to growth on cellulose, including transhydrogenation of NADH to NADPH and increased nitrogen assimilation.
    No preview · Article · Dec 2012 · Canadian Journal of Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The Km values of 201 and 146 μm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.
    Preview · Article · Nov 2012 · Journal of Biological Chemistry
  • Dominic Pinel · Vincent J. J. Martin
    No preview · Article · Oct 2012
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    [Show abstract] [Hide abstract] ABSTRACT: Polyketides are an important group of secondary metabolites, many of which have important industrial applications in the food and pharmaceutical industries. Polyketides are synthesized from one of three classes of enzymes differentiated by their biochemical features and product structure: type I, type II or type III polyketide synthases (PKSs). Plant type III PKS enzymes, which will be the main focus of this review, are relatively small homodimeric proteins that catalyze iterative decarboxylative condensations of malonyl units with a CoA-linked starter molecule. This review will describe the plant type III polyketide synthetic pathway, including the synthesis of chalcones, stilbenes and curcuminoids, as well as recent work on the synthesis of these polyketides in heterologous organisms. The limitations and bottlenecks of heterologous expression as well as attempts at creating diversity through the synthesis of novel "unnatural" polyketides using type III PKSs will also be discussed. Although synthetic production of plant polyketides is still in its infancy, their potential as useful bioactive compounds makes them an extremely interesting area of study.
    Full-text · Article · Oct 2012 · Computational and Structural Biotechnology Journal
  • No preview · Article · Oct 2012 · Genome
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    No preview · Dataset · Aug 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.
    No preview · Article · Jan 2012 · Applied Microbiology and Biotechnology
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    [Show abstract] [Hide abstract] ABSTRACT: Plants display an immense diversity of specialized metabolites, many of which have been important to humanity as medicines, flavors, fragrances, pigments, insecticides and other fine chemicals. Apparently, much of the variation in plant specialized metabolism evolved through events of gene duplications followed by neo- or sub-functionalization. Most of the catalytic diversity of plant enzymes is unexplored since previous biochemical and genomics efforts have focused on a relatively small number of species. Interdisciplinary research in plant genomics, microbial engineering and synthetic biology provides an opportunity to accelerate the discovery of new enzymes. The massive identification, characterization and cataloguing of plant enzymes coupled with their deployment in metabolically optimized microbes provide a high-throughput functional genomics tool and a novel strain engineering pipeline.
    Full-text · Article · Dec 2011 · Trends in Biotechnology

Publication Stats

2k Citations
233.48 Total Impact Points

Institutions

  • 2007-2015
    • Concordia University Montreal
      • • Centre for Structural and Functional Genomics
      • • Department of Biology
      Montréal, Quebec, Canada
  • 2004-2006
    • Lawrence Berkeley National Laboratory
      • Physical Biosciences Division
      Berkeley, California, United States
  • 1999-2006
    • University of British Columbia - Vancouver
      • Department of Microbiology and Immunology
      Vancouver, British Columbia, Canada
  • 2000-2003
    • University of California, Berkeley
      • Department of Bioengineering
      Berkeley, California, United States