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

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 (BsSDR) 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 BsSDR targeting key hotpots was constructed. Four of the constructed 31 variants exhibited markedly enhanced activity. Subsequent combinatorial mutagenesis generated a "best" variant BsSDR_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 BsSDR also possesses a remarkable capacity to utilize the noncanonical cofactor NMNH. Moreover, in addition to robust enantioselective reductive activity toward CFA, the BsSDR also exhibits glucose-oxidizing activity, driving cofactors regeneration. In the presence of NMNH, BsSDR_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 BsSDR_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 BsSDR as a cost-effective and sustainable biocatalyst for the efficient synthesis of (S)-CFL and other high-value chiral alcohols.