Production of 2,3-butanediol from cellulosic biomass by metabolically engineered Saccharomyces cerevisiae

Abstract

2,3-Butanediol (2,3-BD) is a platform chemical with wide industrial applications. Most microbial fermentations for 2,3-BD production have been focused on pathogenic bacteria, which makes large-scale fermentation difficult in terms of safety and industrialization. Since Saccharomyces cerevisiae, a popular GRAS (Generally Recognized As Safe) microorganism, is known to produce a trace amount of 2,3-BD naturally, the bakers yeast was metabolically engineered for efficient production of 2,3-BD by introducing the 2,3-BD metabolic pathways and by modulating the central carbon metabolism. A fed-batch fermentation strategy was optimized in order to enhance a final 2,3-BD concentration. To intensify the 2,3-BD biosynthetic pathway, the alsS gene encoding α-acetolactate synthase and the alsD gene encoding α-acetolactate decarboxylase both from Bacillus subtilis and the endogenous BDH1 gene coding for 2,3-BD dehydrogenase were overexpressed in the wild-type S. cerevisiae (D452-2). The resulting strain of S. cerevisiae BD0 showed approximately a 10-fold increase in 2,3-BD production compared to the wild strain, but still produced unfavorable ethanol as a major metabolite. To increase 2,3-BD production through eliminating ethanol production, a pyruvate decarboxylase (Pdc)-deficient mutant (SOS4) was used as a host for 2,3-BD production. The SOS4 strain grew in a glucose medium and accumulated pyruvate from glucose, a key intermediate for 2,3-BD, without ethanol production. When the alsS and alsD genes from B. subtilis and the endogenous BDH1 gene were overexpressed in the SOS4, the resulting strain (BD4) not only produced 2,3-BD with a high yield of 0.34 g 2,3-BD/g glucose, but also consumed glucose faster than the parental strain. In a fed-batch fermentation under the optimum aeration condition, 2,3-BD concentration increased up to 96.2 g/L from glucose. The use of xylose that is abundant in lignocellulosic hydrolyzate would make the production of 2,3-BD more sustainable and economical. However, S. cerevisiae cannot ferment xylose as a sole carbon source. For xylose utilization, the XYL1, XYL2, and XYL3 genes coding for xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) derived from Scheffersomyces stipitis were introduced into the SOS4 strain. The resulting strain (SOS4X) accumulated pyruvate by using xylose without ethanol production. Additionally, the alsS and alsD genes from B. subtilis and the endogenous BDH1 gene were overexpressed in the SOS4X for production of 2,3-BD from xylose. As a result, the resulting strain (BD4X) produced 20.7 g/L 2,3-BD with a yield of 0.27 g 2,3-BD/g xylose, showing that (R, R)-2,3-BD was dominantly produced. The titer of 2,3-BD from xylose increased up to 43.6 g/L in a fed-batch fermentation. These results suggest that S. cerevisiae might be a promising host for producing 2,3-BD from lignocellulosic biomass for industrial applications.