Abstract
NADPH plays a crucial role in cellular metabolism for biosynthesis and oxidative stress responses. We previously developed the genetically encoded NADPH biosensor pSenSox based on the transcriptional regulator SoxR of Escherichia coli, its target promoter P(soxS) and eYFP as fluorescent reporter. Here, we used pSenSox to study the influence of various parameters on the sensor output in E. coliduring reductive biotransformation of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by the strictly NADPH-dependent alcohol dehydrogenase of Lactobacillus brevis (LbAdh). Redox-cycling drugs such as paraquat and menadione strongly activated the NADPH biosensor and mechanisms responsible for this effect are discussed. Absence of the RsxABCDGE complex and/or RseC caused an enhanced biosensor response, supporting a function as SoxR-reducing system. Absence of the membrane-bound transhydrogenase PntAB caused an increased biosensor response, whereas the lack of the soluble transhydrogenase SthA or of SthA and PntAB was associated with a strongly decreased response. These data support the opposing functions of PntAB in NADP(+) reduction and of SthA in NADPH oxidation. In summary, the NADPH biosensor pSenSox proved to be a useful tool to study NADPH-related processes in E. coli.