Dataset Information

Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering.

ABSTRACT:

Background

2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production.

Results

We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH) pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 . l-1) and yield (0.113 g . g-1) were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition.

Conclusions

With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

SUBMITTER: Ng CY

PROVIDER: S-EPMC3442981 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.