Dataset Information

Improved vanillin production in baker's yeast through in silico design.

ABSTRACT:

Background

Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.

Results

Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin ?-D-glucoside yield in batch mode, while continuous culture of the ?pdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast.

Conclusion

Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

SUBMITTER: Brochado AR

PROVIDER: S-EPMC2992047 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Improved vanillin production in baker's yeast through in silico design.

Brochado Ana Rita AR Matos Claudia C Møller Birger L BL Hansen Jørgen J Mortensen Uffe H UH Patil Kiran Raosaheb KR

Microbial cell factories 20101108

<h4>Background</h4>Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an a ...[more]

PMID: 21059201

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Project description:BackgroundIntegrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells.ResultsUsing constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae.ConclusionsThis study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.

Project description:One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using a two-pronged apparoach: (i) a global proteomic analysis supported by multiple physiological experiments and mutant analyses (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin. Upregulated proteins included enzymes capable of converting aldehydes to potentially less toxic compounds; pentose phosphate pathways enzymes, fumarase C and enzymes of the glyoxylate shunt; proteins involved in several key stress responses, in particular oxidative stress; proteins mediating uptake and processing of metal ions. 1H-NMR confirmed that E. coli detoxifies vanillin by reduction to vanillyl alcohol; the aldehyde reductases YqhD and DkgA were highly upregulated by vanillin and the purified enzymes reduced vanillinin an NADPH dependent manner. Vanillin caused accumulation of reactive oxygen species and activated an oxidative stress response through SoxRS, OxyR and MarA pathways. Slow vanillin dependent copper (II) to copper (I) reduction lead to upregulation of the copA gene and growth in the presence of vanillin was hypersensitive to inhibition by copper ions. RT-PCR and mutant growth data suggested AcrD and AaeAB as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Purifed variant GltA enzymes all showed higher activity due to a lowered Km for oxaloacetate. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including deletion of the efflux pumps eamA, acrA, and acrB, enhancing oxidative stress defences and NADPH production, improving copper homeostasis and increasing citrate synthase activity using variant enzymes.

Dataset Information

Improved vanillin production in baker's yeast through in silico design.

Background

Results

Conclusion

Publications

Improved vanillin production in baker's yeast through in silico design.

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