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 $\textit{Escherichia coli}$ and $\textit{Corynebacterium glutamicum}$ 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 $\textit{Lactobacillus brevis}$. Partial cyclization of the PPP in $\textit{E. coli}$ and $\textit{C. glutamicum}$ was achieved by deletion of the phosphofructokinase gene $\textit{pfkA}$, which prevents fructose 6-phosphate catabolism in the glycolytic pathway. The $\textit{pfkA}$-deficient mutants carrying the $\textit{L. brevis}$ ADH showed a doubled MHB-per-glucose ratio compared to the parent strains. In $\textit{E. coli}$ the partial PPP cyclization in the Δ$\textit{pfkA}$ mutant was proven by $^{13}$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 Δ$\textit{pfkA}$ 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 $\textit{Zymomonas mobilis}$ enhanced the specific MHB productivity by 21% in the $\textit{E. coli ΔpfkA}$ mutant. Deletion of glyceraldehyde 3-phosphate dehydrogenase ($\textit{gapA}$) theoretically results in a completely cyclized PPP and a ratio of 12 mol NADPH per mol glucose 6-phosphate. A $\textit{C. glutamicum ΔgapA}$ 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 $\textit{E. coli}$ during biotransformation showed an upregulation of $\textit{soxS}$ transcription after MAA addition, suggesting that the SoxR regulator known to upregulate soxS expression is activated by a lowered NADPH/NADP$^{+}$ ratio. Subsequently, the $\textit{soxS}$ promoter was fused on a plasmid with the gene encoding yellow fluorescent protein (eYFP). $\textit{E. coli}$ transformed with this plasmid showed fluorescence when MAA was added to the culture. The final fluorescence [...