Michel Schalk

Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants

Terpenes constitute a distinct class of natural products1 that attract insects, defend against phytopathogenic microbes and combat human diseases. However, like most natural products, they are usually made by plants and microbes in small amounts and as complex mixtures. Chemical synthesis is often costly and inefficient, and may not yield enantiomerically pure terpenes, whereas large-scale microbial production requires expensive feedstocks. We engineered high-level terpene production in tobacco plants by diverting carbon flow from cytosolic or plastidic isopentenyl diphosphate through overexpression in either compartment of an avian farnesyl diphosphate synthase and an appropriate terpene synthase. Isotopic labeling studies suggest little, if any, metabolite exchange between these two subcellular compartments. The strategy increased synthesis of the sesquiterpenes patchoulol and amorpha-4,11-diene more than 1,000-fold, as well as the monoterpene limonene 10–30 fold, and seems equally suited to generating higher levels of other terpenes for research, industrial production or therapeutic applications.

Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

BackgroundTerpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.ResultsWe present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.ConclusionsThe highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase encoded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain.

Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism.

Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of α-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.

Enhancement of farnesyl diphosphate pool as direct precursor of sesquiterpenes through metabolic engineering of the mevalonate pathway in Saccharomyces cerevisiae

The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over‐expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down‐regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over‐expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol. Biotechnol. Bioeng. 2010; 106: 86–96.

Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae.

BackgroundSesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for α-santalene production.ResultsA multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall α-santalene yield of a 0.0052 Cmmol (Cmmol glucose)-1 corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final α-santalene productivity of 0.036 Cmmol (g biomass)-1 h-1.ConclusionsThe results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene α-santalene. This represents a starting point toward the construction of a yeast “sesquiterpene factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods.

Toward a Biosynthetic Route to Sclareol and Amber Odorants

Ambergris, a waxy substance excreted by the intestinal tract of the sperm whale, has been a highly prized fragrance ingredient for millenia. Because of supply shortage and price inflation, a number of ambergris substitutes have been developed by the fragrance industry. One of the key olfactory components and most appreciated substitutes of ambergris, Ambrox is produced industrially by semisynthesis from sclareol, a diterpene-diol isolated from Clary sage. In the present study, we report the cloning and functional characterization of the enzymes responsible for the biosynthesis of sclareol. Furthermore, we reconstructed the sclareol biosynthetic pathway in genetically engineered Escherichia coli and reached sclareol titers of ~1.5 g/L in high-cell-density fermentation. Our work provides a basis for the development of an alternative, sustainable, and cost-efficient route to sclareol and other diterpene analogues.

Regulation of the Cinnamate 4-Hydroxylase (CYP73A1) in Jerusalem Artichoke Tubers in Response to Wounding and Chemical Treatments

trans-Cinnamate 4-hydroxylase (C4H) is a plant-specific cytochrome (P450) that is encoded by the gene CYP73A and catalyzes the second step of the multibranched phenylpropanoid pathway. Increases in C4H activity in response to physical and chemical stresses have been well documented, but the mechanism of these increases has never been studied in detail. This paper reports on the regulatory mechanism controlling C4H activity in Jerusalem artichoke (Helianthus tuberosus) tubers in response to wounding and chemical treatments. We compared induction of C4H and other P450-catalyzed activities. C4H was moderately induced by chemicals relative to other P450s. Increases in enzyme activity, C4H protein, and transcripts were quantified and compared in tuber tissue 48 h after wounding and chemical treatments. Our data suggest that induction of the enzyme activity results primarily from gene activation. Time-course experiments were performed after wounding and aminopyrine treatment. Compared with wounded tissues, aminopyrine triggered an additional and delayed peak of transcript accumulation. The timing of the induced changes in activity, protein, and transcripts confirms that C4H induction results primarily from an increase in CYP73A1 mRNA, in both wounded and aminopyrine-treated tissues. However, posttranscriptional mechanisms might also contribute to the regulation of C4H activity.

Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

BackgroundThe need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering.ResultsIn this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function.ConclusionsWith an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.