Dataset Information

Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.

ABSTRACT: Vindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P450 enzymes (CYPs) with appropriate cytochrome P450 reductases (CPRs), engineering the microenvironment for functional expression of CYPs, enhancing cofactor supply, and optimizing fermentation conditions, the production of vindoline was increased to a final titer as high as ∼16.5 mg/L, which is more than 3,800,000-fold higher than the parent strain and the highest tabersonine to vindoline conversion yield ever reported. This work represents a key step of the engineering efforts to establish de novo biosynthetic pathways for vindoline, vinblastine, and vincristine.

SUBMITTER: Liu T

PROVIDER: S-EPMC8446080 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.

Liu Tengfei T Huang Ying Y Jiang Lihong L Dong Chang C Gou Yuanwei Y Lian Jiazhang J

Communications biology 20210916 1

Vindoline is a plant derived monoterpene indole alkaloid (MIA) with potential therapeutic applications and more importantly serves as the precursor to vinblastine and vincristine. To obtain a yeast strain for high yield production of vindoline from tabersonine, multiple metabolic engineering strategies were employed via the CRISPR/Cas9 mediated multiplex genome integration technology in the present study. Through increasing and tuning the copy numbers of the pathway genes, pairing cytochrome P45 ...[more]

PMID: 34531512

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Project description:BackgroundGlycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground.ResultsIn this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with β-AS (β-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of β-amyrin and 14 μg/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630.ConclusionsThis study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.

Project description:Background2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol.ResultsEndogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h.ConclusionsThe microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Dataset Information

Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.

Publications

Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.

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