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 Enterobacter aerogenes (E. aerogenes) by increasing the intracellular NADH pool. Increasing the NADH/NAD+ ratio by knocking out the NADH dehydrogenase genes (nuoC/nuoD) enhanced 2,3-BD production by up to 67% compared with wild-type E. aerogenes. When lactate dehydrogenase (ldh) 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.

Systemic metabolic engineering of Enterobacter aerogenes for efficient 2,3-butanediol production

“…However, less research has been done in recent years to limit the succinate production (Thapa et al 2019 ). Instead, most studies have focused on limiting lactate (Ge et al 2020 ; Wu et al 2021 ; Chu et al 2021 ). Thapa et al attempted to knock out the mdh gene encoding malate dehydrogenase to inhibit succinate production and further enhance 2,3-BDO production and then showed that 2,3-BDO was not significantly enhanced (Thapa et al 2019 ).…”

“…A dynamic tolerance engineering strategy was designed by introducing the deinococcal response regulator DR1558 to improve microbial productivity and relieve metabolic burden. Engineering strategies to improve cellular stress resistance such as adaptive evolution (Huang et al 2023 ; Zhou et al 2023 ), regulatory factor introduction (Wu et al 2021 ), tolerance target screening (Li et al 2023 ; Cámara et al 2022 ), and transport engineering (Mutanda et al 2022 ) are now widely developed. Among them, the introduction of regulatory factors is a useful strategy to improve tolerance to target compounds even in the face of unknown toxicity mechanisms.…”

“…Enterobacter aerogenes is a facultatively anaerobic Gram-negative bacterium (Wu et al 2021 ), which is one of the most popular strains in the study of green bioenergy in recent years. E. aerogenes can naturally produce 2,3-BDO with a broad substrate spectrum and short growth cycle and is appropriate for high-cell-density fermentation (Lu et al 2022 ).…”

“…When Thapa et al modified E. aerogenes by eliminating ldh A and pta , the 2,3-BDO titer in flask cultivation increased 8.11 times above the wild type (Thapa et al 2019 ). Thus, increasing the biosynthesis of 2,3-BDO through restricting pathways that compete for NADH and carbon is a possible option (Wu et al 2021 ).…”

Elucidation of the metabolic mechanism for malate production in Myceliophthora thermophila via 13C metabolic flux analysis

“…Wu et al (33) knocked out the NADH dehydrogenase genes (nuoC/nuoD) to increase NADH pool and knocked out the lactate dehydrogenase (ldh) to reduce other consumption of NADH, their results showed that 2,3-butanediol production increased by 67% and 71.2%, respectively.…”