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

Fatty acyl-coenzyme A (CoA) thioesters are indispensable intermediates in primary metabolism and essential building blocks for biosynthetic pathways yielding fuels, fine chemicals, and pharmaceuticals. However, large-scale production of acyl-CoAs is frequently constrained by the intracellular availability and high cost of CoA, motivating the development of alternative strategies for precursor generation. Here, we report the engineering of the acylCoA ligase AcsA_Pc 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.