Channel-Guided Engineering of Tryptophan Synthase β-Subunit for High-Level Biosynthesis of the Garlic Bioactive Compound S-Allyl-l-Cysteine.

S-Allyl-l-cysteine (SAC) is a bioactive organosulfur compound from aged garlic that has attracted considerable interest for its health-promoting properties. Here, we developed a channel-guided engineering strategy to enhance SAC biosynthesis using the β-subunit of tryptophan synthase (TrpB). Genome mining guided by channel similarity-identified a thermostable Thermotoga maritima TrpB (TmTrpB) as a suitable scaffold with intrinsic activity toward allyl mercaptan. Engineering the tunnel-lining residues yielded a beneficial mutation, TmTrpB4T, which increased SAC production by 2.5-fold compared with the wild-type enzyme. Molecular simulations indicated that this mutation improved substrate accessibility by increasing channel hydrophobicity and optimizing tunnel geometry. The engineered enzyme also exhibited broad substrate promiscuity toward diverse thiols. Under optimized whole-cell biocatalytic conditions, SAC reached 87 g·L-1 with 90% serine conversion. This work demonstrates the utility of channel-guided engineering for improving TrpB catalysis and provides an efficient biocatalytic platform for the synthesis of pharmaceutically valuable S-substituted l-cysteines.