Metabolic Engineering of Enterobacter aerogenes for Improved 2,3-Butanediol Production by Manipulating NADH Levels and Overexpressing the Small RNA RyhB

Abstract

Biotechnological production of 2,3-butanediol (2,3-BD), a versatile platform bio-chemical and a potential biofuel, is limited due to by-product toxicity. In this study, we aimed to redirect the metabolic flux toward 2,3-BD in <i>Enterobacter aerogenes</i> (<i>E. aerogenes</i>) by increasing the intracellular NADH pool. Increasing the NADH/NAD⁺ ratio by knocking out the NADH dehydrogenase genes (<i>nuoC</i>/<i>nuoD</i>) enhanced 2,3-BD production by up to 67% compared with wild-type <i>E. aerogenes</i>. When lactate dehydrogenase (<i>ldh</i>) was knocked out, the yield of 2,3-BD was increased by 71.2% compared to the wild type. Metabolic flux analysis revealed that upregulated expression of the sRNA RyhB led to a noteworthy shift in metabolism. The 2,3-BD titer of the best mutant Ea-2 was almost seven times higher than that of the parent strain in a 5-L fermenter. In this study, an effective metabolic engineering strategy for improved 2,3-BD production was implemented by increasing the NADH/NAD⁺ ratio and blocking competing pathways.