Aldolases, especially 2-deoxyribose-5-phosphate aldolase (DERA) enzymes, have been widely employed to access key chiral precursors for various active pharmaceutical ingredients (APIs). This has been enabled by expanding their substrate scope toward non-natural acceptors and donors via protein engineering. In this study, we endeavored to broaden the acceptor substrate scope of DERA from Geobacillus sp. (DERAGeo) toward the heteroaryl aldehydes through a rational protein engineering approach. We successfully performed iterative saturation mutagenesis of DERAGeo, resulting in two enantiocomplementary variants, viz., (R)-selective DERAGeo-S185G and (S)-selective DERAGeo-T12I/S185A, with enhanced catalytic efficiencies and enantioselectivities. Remarkably, the natural enantioselectivity of DERAGeo was reversed by a single mutation (S185G). The synthetic applicability of the mutants was demonstrated by conducting aldol reactions on a semipreparative scale, from which both (R)- and (S)-enantiomers of heteroaryl aldols were isolated with high yields (up to 99%) and excellent enantiopurities (up to 99:1 e.r.).
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