Lin Yuan,Yan-Li Qin,Zhi-Cheng Zou,Bright Appiah,Hao Huang,Zhong-Hua Yang,Can Qun
The intracellular NAD(P)H insufficiency is the key factor which limits the reduced product (such as chiral alcohols) synthesis by whole cell biocatalysis or microbial cell factory. In this paper, we reported a novel solution to increase NADPH supply through strengthening the pentose phosphate pathway (PPP) flux with overexpression of extra zwf (gene for glucose 6-phosphatedehydrogenase) and glk (gene for glucokinase) by recombinant Escherichia coli BL21(DE3)/pETDuet-1- glk - zwf and pET28a- bccr containing a carbonyl reductase gene bccr . The amount of intracellular NADPH was significantly increased from 150.3 μmol/L to 681.8 μmol/L after strengthening the PPP flux, which was 4.5-fold to that of the control. It was applied to improve the asymmetric reduction of 4-chloroacetoacetate to ethyl S -4-chloro-3-hydroxybutylate catalyzed by the Bc CR, which increased the reaction yield 2.8-fold to the control. This strategy provides a new way to increase NADPH supply in E. coli cell factories.
In our previous study, a whole-cell biocatalytic system involving α-glucan phosphorylase , phosphoglucomutase , glucose 6-phosphate isomerase, tagatose 1,6-bisphosphate aldolase , and phosphoglycolate phosphatase , was constructed to produce d -tagatose from maltodextrin . However, the biotransformation efficiency of this whole-cell catalyst was low because of the unbalanced ratio of each enzyme and metabolic flux of the intermediates. Therefore, in this study, the biosynthesis of d -tagatose was enhanced by optimizing vectors and improving the expression of rate-limiting enzymes through the construction of multi-copy genes to regulate expression levels. The conversion rate increased from 20.8% after 24 h to 25.2% after 3 h using 10 g/L maltodextrin as the substrate. Furthermore, the genes in the bypass pathways in Escherichia coli , pfka and zwf , were deleted to increase the accumulation of intermediates. The strain ER-2GatZ (Δ p Δ z ) produced 3.383 g/L d -tagatose using 10 g/L substrate after 3 h, which was 1.34 times that of the wild strain. This work exemplifies the use of modular engineering to enhance d -tagatose production using whole-cell catalysts.
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