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

Alina Spielmann,Meike Baumgart,Michael Bott,Hugo Van Beek,Lea Sundermeyer,Yannik Brack,Lion Flachbart

doi:10.1186/s13568-020-0946-7

Alina Spielmann, Meike Baumgart + Show 5 more

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https://doi.org/10.1186/s13568-020-0946-7

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Journal: AMB Express	Publication Date: Jan 18, 2020
Citations: 4	License type: open-access

Affiliation: Forschungszentrum Jülich

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 × 106 clones created by random mutagenesis we identified the variant LbADHK71E. Kinetic analysis of the purified enzyme revealed that LbADHK71E had a ~ 16% lowered KM value and a 17% higher Vmax 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.

Highlights

Chiral alcohols with high enantiomeric purity are important intermediates for the synthesis of optically active fine chemicals that are used for example to produce pharmaceuticals and agrochemicals (Ager et al 1996; Liese et al 2006; Breuer et al 2004)
Construction of an alcohol dehydrogenase of Lactobacillus brevis (LbADH) library and fluorescence-activated cell sorting (FACS) screening We aimed at finding LbADH variants with improved catalytic properties for the reduction of 2,5–hexandione to (2R,5R)-hexanediol by FACS-based HT-screening using the NADPH biosensor pSenSox
A culture expressing the LbadhLibrary was used for the biotransformation of 2,5-hexanedione by incubation for 2.5 h in 2xTY medium supplemented with 70 mM of the diketone

Summary

Introduction

Chiral alcohols with high enantiomeric purity are important intermediates for the synthesis of optically active fine chemicals that are used for example to produce pharmaceuticals and agrochemicals (Ager et al 1996; Liese et al 2006; Breuer et al 2004). LbADH is active as a homotetramer with 251 amino acid residues and a molecular mass of 26.6 kDa per subunit (Riebel 1997; Niefind et al 2003). It is a short-chain Mg2+-dependent reductase that uses the NADPH as cofactor. The non-covalently bound cofactor NADPH is essential for catalysis and must be recycled efficiently to make the biotransformation economically feasible (Leuchs and Greiner 2011; Döbber et al 2018)

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