Engineering of CoA‐Acylating Butyraldehyde Dehydrogenase for Enhanced 1,3‐Butanediol Production in Escherichia coli

Microbial production of 1,3-butanediol (1,3-BDO) offers a renewable route to this versatile C₄ chemical. However, the low performance of CoA-acylating butyraldehyde dehydrogenase (Bld), which contains a catalytic cysteine, limits efficient production in recombinant Escherichia coli (E. coli). In this study, wild-type Clostridium saccharoperbutylacetonicum Bld and its variant Bld* were biochemically characterized and engineered to improve conversion of 3-hydroxybutyryl-CoA (3-HB-CoA) to 3-hydroxybutyraldehyde (3-HBA). Enzyme activity was strongly reduced by the product, 3-HBA, and this reduction was largely alleviated by added cysteine. To mitigate this interference, several noncatalytic cysteine residues in Bld* were substituted individually and in combination guided by multiple sequence alignment and machine-learning-based mutational prediction. The triple mutant C151N/C189A/C353L (designated CYS31) displayed ~30% higher specific activity without altering substrate affinity or selectivity. Incorporation of CYS31 into a 1,3-BDO-producing E. coli strain led to a corresponding ~30% increase in titer, indicating that enhanced in vitro kinetics translated to higher in vivo 1,3-BDO production. These findings provide a more effective Bld variant for 1,3-BDO production and demonstrate that non-active-site cysteine residues can be viable engineering targets when an aldehyde intermediate is involved.