Background: 2 0 -Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5 0 -diphosphate (GDP)-L-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP-L-fucose biosynthetic enzymes and heterologous fucosyltransferase.
Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses. This review summarizes several metabolic engineering approaches used for improving the NADPH regenerating capacity in engineered E. coli for whole-cell-based bioprocesses and discusses the key features and progress of those attempts.
Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.
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