Highly Efficient One-Pot Multienzyme Cascades for the Synthesis of S-Allyl-l-Cysteine from l-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 in vitro multienzyme cascade catalysis system comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase B (OASS B) was established using l-serine and allyl mercaptan as substrates for the efficient synthesis of SAC. Three SAT mutants (SATT167A, SATM201R, and SATN264X) were designed to weaken the feedback inhibitory effect of SAC on SAT activity, with one SATM201R 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 in vitro. This study provides an alternative economical and environmentally sustainable biosynthetic pathway for the efficient production of SAC.