Highly Efficient One-Pot Multienzyme Cascades for the Synthesis of S-Allyl-<scp>l</scp>-Cysteine from <scp>l</scp>-Serine and Allyl Mercaptan

S-Allyl-l-cysteine (SAC) is an important bioactive substance in garlic, which has potential preventive and therapeutic effects, including antioxidant, anticancer, hepatoprotective, and neuroprotective properties. Here, a promising <i>in vitro</i> multienzyme cascade catalysis system comprising serine acetyltransferase (SAT) and <i>O</i>-acetylserine sulfhydrylase B (OASS B) was established using l-serine and allyl mercaptan as substrates for the efficient synthesis of SAC. Three SAT mutants (SAT^T167A, SAT^M201R, and SAT^N264X) were designed to weaken the feedback inhibitory effect of SAC on SAT activity, with one SAT^M201R mutant achieving a SAC yield of 75.5%. Furthermore, by coupling an acetyl-CoA regeneration system, we improved the synthetic yield of the SAC cascade catalytic system to 76.9%, enabling a cost-effective and efficient one-pot enzymatic synthesis of SAC <i>in vitro</i>. This study provides an alternative economical and environmentally sustainable biosynthetic pathway for the efficient production of SAC.