Alcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 10 6 clones created by random mutagenesis we identified the variant LbADH K71E. Kinetic analysis of the purified enzyme revealed that LbADH K71E had a ~ 16% lowered K M value and a 17% higher V max for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.

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. coli during reductive biotransformation of methyl acetoacetate (MAA) to ( R )‐methyl 3‐hydroxybutyrate (MHB) by the strictly NADPH‐dependent alcohol dehydrogenase of Lactobacillus brevis ( Lb Adh). 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 .