Metabolic Engineering of Corynebacterium Glutamicum for Efficient Production of Optically Pure (2R,3R)-2,3-Butanediol

Abstract

Background:2,3-butanediol is an important platform compound which has a wide range of applications, involving in medicine, chemical industry, food and other fields. Especially the optically pure (2R,3R)-2,3-butanediol can be employed as an antifreeze agent and as the precursor for producing chiral compounds. However, some (2R,3R)-2,3-butanediol overproducing strains are pathogenic such as Enterobacter cloacae and Klebsiella oxytoca.Results: In this study, a (3R)-acetoin overproducing C. glutamicum strain, CGS9, was engineered to produce optically pure (2R,3R)-2,3-butanediol efficiently. Firstly, the gene bdhA from B. subtilis 168 was integrated into strain CGS9 and its expression level was further enhanced by using a strong promoter Psod and ribosome binding site (RBS) with high translation initiation rate, and the (2R,3R)-2,3-butanediol titer of the resulting strain was increased by 33.9%. Then the transhydrogenase gene udhA from E.coli was expressed to provide more NADH for 2,3-butanediol synthesis, which reduced the accumulation of the main byproduct acetoin by 57.2%. Next, a mutant atpG was integrated into strain CGK3, which increased the glucose consumption rate by 10.5% and the 2,3-butanediol productivity by 10.9% in shake-flask fermentation. Through fermentation engineering, the most promising strain CGK4 produced a titer of 144.9 g/L (2R,3R)-2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h in fed-batch fermentation. The optical purity of the resulting (2R,3R)-2,3-butanediol surpassed 99%.Conclusion: To the best of our knowledge, this is the highest titer of optically pure (2R,3R)-2,3-butanediol achieved by GRAS strains, and the result has demonstrated that C. glutamicum is a competitive candidate for (2R,3R)-2,3-butanediol production.