The enzymatic transamination of ketones into (R)amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of (R)-selective amine transaminases [(R)-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an (R)-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the D-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg −1 in the conversion of (R)-phenylethylamine and pyruvate to acetophenone and D-alanine. This value is similar to those of natural (R)-ATAs, which typically are in the range of 250 milliunits mg −1. These results demonstrate that (R)-ATAs can evolve from α-AAT as shown here for the DATA scaffold.

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The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.

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