ABSTRACT: Background:2,3-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. Results:Endogenous 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. Conclusions:The 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.
