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Biosynthesis of guanosine 5′-diphosphate- l-fucose (GDP- l-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP- l-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP- l-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP- l-fucose production. However, GDP- l-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP- l-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP- l-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP- l-fucose concentration of 235.2 ± 3.3 mg l, corresponding to a 21% enhancement in the GDP- l-fucose production compared with the control strain overexpressing GDP- l-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP- l-fucose production in recombinant E. coli. [ABSTRACT FROM AUTHOR]

Whole-cell conversion of cyclohexanone to ɛ-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and ɛ-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to ɛ-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of ɛ-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 ɛ-caprolactone concentration of 11.0 g/l. The maximum ɛ-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 ɛ-caprolactone concentration compared with the control experiment performed under the same conditions. [ABSTRACT FROM AUTHOR]

A recombinant Escherichia coli strain was developed to produce guanosine 5′-diphosphate (GDP)- l-fucose, donor of l-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP- d-mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP- l-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25°C and 0.1 mM isopropyl-β- d-thioglucopyranoside. Maximum GDP- l-fucose concentration of 38.9 ± 0.6 mg l−1 was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 ± 0.5 mg l−1 GDP- l-fucose under the same cultivation condition. [ABSTRACT FROM AUTHOR]

Coenzyme Q10 (CoQ10) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ10 has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ10 with isopentenyl diphosphate. TheddsAgene coding for DPS fromGluconobacter suboxydanswas expressed under the control of anEscherichia coliconstitutive promoter. Analysis of the cell extract in recombinantE. coliBL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ10 rather than endogenous CoQ8 was biologically synthesized as the major coenzyme Q. Expression of theddsAgene with low copy number led to the accumulation of CoQ10 to 0.97 mg l-1 in batch fermentation. A high cell density (103 g l-1) in fed-batch fermentation ofE. coliBL21/pACDdsA increased the CoQ10 concentration to 25.5 mg l-1 and its productivity to 0.67 mg l-1 h-1, which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation. [ABSTRACT FROM AUTHOR]