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Free keywords:
directed evolution; semirational design; iterative saturation mutagenesis; stereoselectivity; regioselectivity; thermostability
Abstract:
In the quest to produce by directed evolution chemo-, stereo-, and regioselective enzymes as catalysts for the sustainable production of chiral compounds needed in modern society, focused saturation mutagenesis at sites surrounding the binding pocket has emerged as a particularly effective strategy. This chapter focuses on the genesis and recent progress of this concept, which has been dubbed combinatorial active-site saturation test (CAST), a procedure that can be performed iteratively (iterative saturation mutagenesis, ISM). The newest developments aimed at increasing efficacy further include guidelines on how to choose hotspots for CAST and how to design reduced amino acid alphabets semirationally, multiparameter evolution aided by mutability landscapes, utilization of the CRISP-Cas9 system, and solid-phase chemical syntheses of saturation mutagenesis libraries on Si-chips for eliminating amino acid bias. The chapter includes a table of recent CAST/ISM papers and highlights in detail three case studies: Limonene epoxide hydrolase in desymmetrization of a prochiral epoxide, alcohol dehydrogenase (TbSADH)-catalyzed enantioselective transformation of difficult-to-reduce ketones, and P450-BM3 as a biocatalyst in whole-cell cascade reactions of cyclohexane with production of ( R,R )-, ( S,S )-, and ( R,S )-1,2-dihydroxycyclohexane.
