Engineering an Acyl‐CoA Ligase With Enhanced Activity Toward Synthetic CoA Alternatives

to preferentially utilize inexpensive, membrane-permeable synthetic CoA surrogates, with a focus on N-acetylcysteamine (SNAC). Building on a broad specificity variant (D449E), structure-guided saturation mutagenesis and random mutagenesis, coupled with high-throughput colorimetric screening, progressively shifted thiol specificity from CoA to pantetheine and SNAC. The resulting double mutant, F430W/D449E, exhibits a 26-fold improvement in SNAC utilization relative to wildtype while concomitantly reducing CoA activity. Kinetic analyses reveal that these gains arise from increased reaction rates with synthetic thiols and altered substrate preferences across a representative acid panel. The functional relevance of AcsA-generated acyl-SNACs was demonstrated in a reconstituted polyketide system, where enhanced availability of SNAC-linked starter units drove an ∼8-fold increase in pyrone formation. This work establishes AcsA as a tunable platform for orthogonal generation of polyketide precursors and a general framework for alleviating CoA-dependent biosynthetic bottlenecks.