Enhanced 2,3-Butanediol Production in Recombinant Klebsiella pneumoniae via Overexpression of Synthesis-Related Genes

Abstract

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.

Industrial Production of 2,3-Butanediol from the Engineered Corynebacterium glutamicum

“…A significant decrease in 2,3-BDO production by SGSC103 and SGSC104 can be explained by budC overexpression, which has a negative effect to 2,3-BDO biosynthesis in C. glutamicum. The acetoin reductase encoded by budC in K. pneumoniae also participates in a reverse reaction that converts 2,3-BDO to acetoin, as has been demonstrated in previous studies [17]. Therefore, we suppose that the introduction of acetoin reductase from K. pneumoniae disturbed the 2,3-BDO synthesis by facilitating the reverse reaction at the physiological conditions of C. glutamicum.…”

Construction of nitrogen-fixingKlebsiella variicolaGN02 expression vector pET28a-Lac-EGFP and its colonization ofPennisetum giganteumz.x.lin roots

“…Klebsiella variicola GN02, isolated from the roots of mature P. sinense, is an endogenous bacterium with high nitrogen-fixing, phosphorus-dissolving, ammoniaproducing, iron carrier-producing and antibiotic-promoting properties and, therefore, is a good strain source for fertilizers [16]. K. variicola has few available vectors and the current major vector used in transformation experiments of Klebsiella-related strains is pET28a [17]. This is a widely used prokaryotic expression vector [18] because it can be expressed in gramnegative bacteria.…”

A non-pathogenic and optically high concentrated (R,R)-2,3-butanediol biosynthesizing Klebsiella strain

“…Thus, the carbon flux toward (R,R)-2,3-BDO biosynthesis and (R,R)-2,3-BDO biosynthesis was increased because of overexpression of the GldA and DhaD genes. In Klebsiella species, 2,3-BDO synthesis plays a role in regulating the intracellular NAD + balance by consuming NADH (Jung et al, 2012;Kim et al, 2012). As shown in Fig.…”

“…As the biosynthetic pathways for 2,3-BDO and lactic acid both consume NADH, these pathways compete with each other. Lactate synthesis from pyruvate consumes 1 mole of NADH per 1 mol of pyruvate, thus NADH spared from being utilized in the lactate pathway by the deletion of the ldhA gene can be used in 2,3-BDO production (Jung et al, 2012;Kim et al, 2012), which explains the enhanced carbon flux toward 2,3-BDO synthesis in the SGSB112 strain.…”