The application of ketoreductase-based biocatalytic reduction to access optically pure Prelog or anti-Prelog alcohols offers a valuable approach for asymmetric synthesis. Despite this, control of the stereopreferences of ketoreductases as desired remains challenging, since natural ketoreductases usually display Prelog preference and it is difficult to transfer the knowledge from engineered anti-Prelog ketoreductases to the others. Here, we present the discovery of a switch between Prelog and anti-Prelog reduction toward halogen-substituted acetophenones in six short-chain dehydrogenase/reductases (SDRs). Through carefully analysis of the structural information and multiple-sequence alignment of several reported SDRs with Prelog or anti-Prelog stereopreference, the key residues that might control their stereopreferences were identified using Lactobacillus fermentum short-chain dehydrogenase/reductase 1 (LfSDR1) as the starting enzyme. Protein engineering at these positions of LfSDR1 could improve its anti-Prelog stereoselectivity or switch its stereopreference to Prelog. Moreover, the knowledge obtained from LfSDR1 could be further transferred to the five other SDRs (four mined SDRs and one reported SDR) that have 21−48% sequence identities with LfSDR1. The stereopreferences of these SDRs were able to be switched either from anti-Prelog to Prelog or from Prelog to anti-Prelog by mutagenesis at related positions. In addition, further optimization of LfSDR1 can access stereocomplementary reduction of several halogen-substituted acetophenones with high stereoselectivity (up to >99%), resulting in some valuable chiral alcohols for the synthesis of pharmaceutical agents.
Enantiopure halohydrins, which are important building blocks for pharmaceutical agents, could be synthesized by biocatalytic reduction of α-halo ketones using ketoreductases. In this study, Candida glabrata ketoreductase 1 (CgKR1) variants with >99% stereoselectivity toward α-halo ketones, such as 2-chloroacetophenone, 2-chloro-4′-fluoroacetophenone, and 2-bromoacetophenone, were obtained through engineering of CgKR1 at residues Phe92 and Tyr208. Interestingly, asymmetric reduction of these α-halo ketones by all the variants of CgKR1 followed anti-Prelog's rule, which is rarely found in natural ketoreductases. Moreover, the biocatalytic processes for reduction of these aromatic α-halo ketones with high substrate loading were achieved by coexpression of glucose dehydrogenase (GDH) for NADPH regeneration, indicating the potential of practical applications of these variants.
Chiral carveol and dihydrocarveol are important additives in the flavor industry and building blocks in the synthesis of natural products. Despite the remarkable progress in asymmetric catalysis, convenient access to all possible stereoisomers of carveol and dihydrocarveol remains a challenge. Here, we present the stereodivergent synthesis of carveol and dihydrocarveol through ketoreductases/ene‐reductases catalyzed asymmetric reduction. By directly asymmetric reduction of (R)‐ and (S)‐carvone using ketoreductases, which have Prelog or anti‐Prelog stereopreference, all four possible stereoisomers of carveol with medium to high diastereomeric excesses (up to >99 %) were first observed. Then four stereoisomers of dihydrocarvone were prepared through ene‐reductases catalyzed diastereoselective synthesis. Asymmetric reduction of obtained dihydrocarvone isomers by ketoreductases further provide access to all eight stereoisomeric dihydrocarveol with up to 95 % de values. In addition, the absolute configurations of dihydrocarveol stereoisomers were determined by using modified Mosher's method.
Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion‐based dual fluorescence assay (SDFA) for high‐throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197‐fold higher kcat/km value toward (S)‐2‐octanol compared to its wild type.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.