Increasing the NADPH supply for whole-cell biotransformation and development of a novel biosensor

Abstract

In the first part of this work, the pentose phosphate pathway (PPP) was investigated as a source of NADPH in reductive whole-cell biotransformation using Escherichia coli\textit{Escherichia coli} and Corynebacterium glutamicum\textit{Corynebacterium glutamicum} as hosts and glucose as reductant. The reduction of methyl acetoacetate to the chiral (R)-methyl hydroxybutyrate (MHB) served as a model reaction for NADPH-dependent reactions and was catalyzed by an alcohol dehydrogenase (ADH) from Lactobacillus brevis\textit{Lactobacillus brevis}. Partial cyclization of the PPP in E. coli\textit{E. coli} and C. glutamicum\textit{C. glutamicum} was achieved by deletion of the phosphofructokinase gene pfkA\textit{pfkA}, which prevents fructose 6-phosphate catabolism in the glycolytic pathway. The pfkA\textit{pfkA}-deficient mutants carrying the L. brevis\textit{L. brevis} ADH showed a doubled MHB-per-glucose ratio compared to the parent strains. In E. coli\textit{E. coli} the partial PPP cyclization in the ΔpfkA\textit{pfkA} mutant was proven by 13^{13}C-flux analysis, which showed a negative net flux through the phosphoglucose isomerase reaction. Furthermore, the flux through pyruvate kinase was found to be absent in the ΔpfkA\textit{pfkA} mutant, indicating that a low phosphoenolpyruvate (PEP) concentration limited glucose uptake via the phosphotransferase system (PTS). PTS-independent glucose uptake and phosphorylation via the glucose facilitator and glucose kinase from Zymomonas mobilis\textit{Zymomonas mobilis} enhanced the specific MHB productivity by 21% in the E. coli ΔpfkA\textit{E. coli ΔpfkA} mutant. Deletion of glyceraldehyde 3-phosphate dehydrogenase (gapA\textit{gapA}) theoretically results in a completely cyclized PPP and a ratio of 12 mol NADPH per mol glucose 6-phosphate. A C. glutamicum ΔgapA\textit{C. glutamicum ΔgapA} mutant showed a ratio of 7.9 mol MHB per mol glucose, which is the highest one reported so far. Formation of the by-product glycerol presumably was responsible for not achieving a higher ratio. In the second part of this work, a biosensor was developed which is capable of detecting a lowered intracellular NADPH/NADP+ ratio and trigger the synthesis of an autofluorescent protein. DNA microarray analysis of E. coli\textit{E. coli} during biotransformation showed an upregulation of soxS\textit{soxS} transcription after MAA addition, suggesting that the SoxR regulator known to upregulate soxS expression is activated by a lowered NADPH/NADP+^{+} ratio. Subsequently, the soxS\textit{soxS} promoter was fused on a plasmid with the gene encoding yellow fluorescent protein (eYFP). E. coli\textit{E. coli} transformed with this plasmid showed fluorescence when MAA was added to the culture. The final fluorescence [...

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