Synthesis of l-cysteine from glycine via a multi-enzyme cascade

L-Cysteine, a crucial amino acid containing an active thiol group, finds extensive applications across various industries such as pharmaceuticals, food, feed, and cosmetics. Despite its significance, the efficient production of l-cysteine has long been a challenging task. This study analyzed the l-cysteine synthesis pathway in microorganisms and designed a novel synthetic route for l-cysteine utilizing glycine as a primary substrate. By employing a cell-free synthetic system catalyzed by multi-enzyme cascades, efficient synthesis of l-cysteine was achieved. The study also implemented a high-throughput screening approach for identifying rate-limiting enzymes using a l-cysteine biosensor, leading to a notable 20.12 % enhancement in the activity of the key rate-limiting enzyme, L‑serine acetyltransferase. Furthermore, through the optimization of catalytic reaction conditions, the l-cysteine titer increased by 2.06-fold, reaching a peak of 19.44 mg/L. This innovative approach utilizing a multi-enzyme cascade pathway unveils promising new avenues for l-cysteine production.