Production of 2,3-Butanediol By Engineered Saccharomyces cerevisiae

2,3-Butanediol (2,3-BD) is a platform chemical with wide applications including a precursor chemical to 1,2-butadiene, a monomer for synthetic rubber. Saccharomyces cerevisiae is an attractive host strain for the production of 2,3-BD. While S. cerevisiae is known to produce trace amounts of 2,3-BD naturally, it is necessary to engineer S. cerevisiae to produce 2,3-BD with high yield and productivity. To increase 2,3-BD production through elimination of ethanol production, a pyruvate decarboxylase (Pdc)-deficient mutant was constructed. Also the alsS and alsD encoding acetolactate synthase and acetolactate decarboxylase from Bacillus subtilis and endogenous BDH coding for 2,3-BD dehydrogenase were overexpressed. The engineered Pdc-deficient S. cerevisiae with the 2,3-BD biosynthetic pathway has two main problems for efficient production of 2,3-BD: redox imbalance induced by accumulation of cytosolic NADH, shortage of C₂-compounds for cell growth. To overcome these problems, NADH oxidase from Lactococcuslactis(noxE) was introduced and the expression levels of PDC were optimized by combination of heterologous PDC genes and native promoters. The reduced intracellular NADH/NAD⁺ ratio by the expression of noxEincreased 2,3-BD yield and decreased glycerol yield as a byproduct. In addition, the optimized expression of PDC relieved C₂-auxotropy and increased 2,3-BD productivity. Through the fed-batch fermentation with the BD5_Ctnox strain with co-expression of noxEand optimized PDC, 154.3 g/L of 2,3-BD was produced with 1.98 g/L/h of productivity. The overall yield of 2,3-BD was 0.404 g/g which corresponds to 80.7% of theoretical yield. The metabolic engineering strategy used in this study will be applicable to production of 2,3-BD as well as other chemicals by using Pdc-deficient S. cerevisiae as a host strain.