A growth selection system for the directed evolution of amine-forming or converting enzymes

Abstract

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.

Computer-Aided Tunnel Engineering: A Promising Strategy for Improving Lipase Applications in Esterification Reactions

“…Structural modeling and molecular docking revealed that M226L opened the substrate tunnel and therefore increased its activity. 96 Similarly, after random and 19 °C and doubled the tolerance to DMSO. Mutagenesis in the tunnel affects the pathway of cosolvent molecules into the active site cavity, and the introduced substitutions close the tunnel and may prevent DMSO molecules from entering the active site and causing structural instability of the protein.…”

Structure‐ and Data‐Driven Protein Engineering of Transaminases for Improving Activity and Stereoselectivity

“…[5] So far, the most effective method of modifying substrate promiscuity, catalytic activity, and stereoselectivity of ATAs is to mutate crucial residues adjacent to the two binding pockets by protein engineering to alter their steric and electronic environment. [6] Recently, a series of rationaldesign or screening methods, such as structure-dependent rational design, [7] mechanism-guided computational design [8] and a growth selection [9] method, have been successfully applied to engineer ATAs for higher activity, broader substrate scope and improved stereoselectivity towards particular substrates. However, these strategies require a significant amount of computational and/or experimental effort to optimize the biocatalyst.…”

Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

“…Ultrahighthroughput screening methods based on a variety of in vitro microscale (i.e. single-cell) compartmentalization 17 strategies or alternatively growth based selections 18,19 enable large library screening. However, these approaches may suffer from limitations such as requiring tailoring for a specific enzyme chemistry, necessitating microfluidic device design, fabrication, and utilization, or otherwise being limited in chemical scope.…”