An Artificial Biosynthetic Pathway for l -Cysteine Mimicking 2-Methylcitrate Cycle

l-Cysteine is a high-value sulfur-containing amino acid indispensable for industrial and medical applications. However, the production of l-cysteine is limited by the low sulfur assimilation efficiency and complex regulatory networks of native pathways. Here, we report the design and construction of a non-native biosynthetic pathway for l-cysteine in Escherichia coli, utilizing 3-mercaptopropionic acid as a starting substrate. Inspired by the 2-methylcitrate cycle, this pathway introduces sulfur via a thiol-functionalized carbon backbone, bypassing the energy-intensive sulfur assimilation pathway and establishing a self-sustaining C4 cycle to preserve central carbon flux. We systematically screened enzymes for the pathway, identifying a Salmonella typhimurium 2-methylcitrate synthase (StPrpC) capable of condensing the thiol-functionalized substrate (3-mercaptopropionyl-CoA), and we enhanced its specificity toward the thio-substrate through structure-guided engineering. The resulting variant, StPrpCD325 K, exhibited a 17.7% reduction in Km (from 23.7 to 19.5 mM) and a 33.1% increase in kcat (from 0.052 to 0.069 s–1), which significantly improved catalytic efficiency toward thio-substrate. Whole-cell biocatalysis using the engineered strain produced 0.63 g/L of l-cysteine with a yield of 0.59 g/g after metabolic optimization, including the deletion of degradation pathways and overexpression of transporters. This work demonstrates an atom-economical strategy for amino acid production and offers a promising synthetic route to alleviate the constraints of traditional fermentation processes.