Engineering a Novel Cofactor‐Versatile Short‐Chain Dehydrogenase for Efficient Synthesis of Chiral Alcohols

ABSTRACT Short‐chain dehydrogenases/reductases (SDRs) are widely used for the asymmetric synthesis of chiral alcohols, which are critical building blocks in pharmaceutical manufacturing. In this study, a novel SDR from Bacillus subtilis ( Bs SDR) was identified and engineered for the asymmetric synthesis of ( S )−1‐(2,6‐dichloro‐3‐fluorophenyl)ethanol (( S )‐CFL). By integrating in silico molecular docking with FuncLib based design, a focused mutant library of Bs SDR targeting key hotpots was constructed. Four of the constructed 31 variants exhibited markedly enhanced activity. Subsequent combinatorial mutagenesis generated a “best” variant Bs SDR M2 (I178L/T179L), which displayed a 107.4‐fold increase in catalytic activity, along with excellent enantioselectivity (> 99.9% e.e .) and broad substrate scope. Notably, unlike most SDRs that are strictly dependent on NAD(H) or NADP(H), the Bs SDR also possesses a remarkable capacity to utilize the noncanonical cofactor NMNH. Moreover, in addition to robust enantioselective reductive activity toward CFA, the Bs SDR also exhibits glucose‐oxidizing activity, driving cofactors regeneration. In the presence of NMNH, Bs SDR M2 displayed a 33.3‐fold improvement in catalytic efficiency ( k cat / K M ) toward substrate 1‐(2,6‐dichloro‐3‐fluorophenyl)ethanone (CFA) compared to the wild‐type (WT), along with an extended half‐life ( t 1/2 ) of 52.1 h at 45°C. Using Escherichia coli expressing Bs SDR M2 as the only biocatalyst and glucose as the cosubstrate, 100 g/L CFA was reduced within 6 h in 92.1% conversion and > 99.9% e.e ., achieving a space‐time yield (STY) of 368.2 g/(L·d). This work highlights the potential of Bs SDR as a cost‐effective and sustainable biocatalyst for the efficient synthesis of ( S )‐CFL and other high‐value chiral alcohols.