Widely applicable background depletion step enables transaminase evolution through solid-phase screening

Chemical Science
Matteo PlanchestainerFrancesca Paradisi

Abstract

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.

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Citations

Aug 15, 2020·Frontiers in Bioengineering and Biotechnology·Christian M HeckmannFrancesca Paradisi
Nov 5, 2019·Frontiers in Bioengineering and Biotechnology·David Roura PadrosaFrancesca Paradisi
Jan 31, 2021·Biotechnology Advances·Pei XuWen-Yong Lou
Jun 25, 2021·Chemical Science·Runkun ZhangGongke Li

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BETA
PCR

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DALI

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