NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

Abstract

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.

Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

“…In the course of these developments, we expected that fusing oligopeptides consisting of TEV protease cleavage sequence (TEVcs, ENLYFQS) (used in the previous study for the BRET application [ 16 ] ) with charged residues (K or D) to the C‐terminus of picALuc might promote intraenzymatic stabilization or regulate luminescence reaction (Figure 1), because a variety of electrostatic interactions (or charge engineering) have been utilized in protein structure and function. [ 17–19 ] Unexpectedly, an increase in luminescence was observed when the reaction was driven by the addition of substrate CTZ h .…”

Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst

“…High-throughput screening methods are essential to the enzyme engineering workflow. Compared to other methods using 96 well plates, , microfluidics, , and fluorescence sorting based assays, cell growth-based selections are more facile and scalable because they utilize cell survival as an easy readout. Furthermore, the selected enzyme variants are directly compatible with in vivo applications.…”