Metabolic Reprogramming in <i>Escherichia coli</i> for Efficient <scp>l</scp>-Cysteine Production by Metabolic Node Engineering

Synthetic biotechnology has boosted the manufacture of biobased chemicals. However, the development of sustainable synthetic routes for compounds with complex metabolic pathways remains challenging. In this study, we propose a metabolic node engineering approach to reprogram Escherichia coli for l-cysteine biosynthesis. By dissection of the metabolic module into input, process, and output nodes, a systematic optimization of key components in l-cysteine production was achieved. First, the input node was redirected by modifying the glucose utilization pathway, expanding the carbon supply pool. Subsequently, the process nodes glycerone phosphate and O-acetyl-l-serine were improved by rational metabolic engineering to enhance synthetic flux and block branched pathways. Furthermore, the l-cysteine output efficiency was enhanced by the construction of a dynamic efflux channel, which served to minimize the overflow of l-serine. Finally, the engineered strain EC21-1/pEX5 produced 20.21 g/L l-cysteine in a 5-L bioreactor. This study provides a systematic method for optimizing biobased chemical synthesis, demonstrating the potential of biotechnology in environmental and resource conservation.