Biosensor-driven Evolution and Transcriptome-Guided Chassis Engineering Enhance Endogenous l -Cysteine Supply for Ergothioneine Production

Abstract Ergothioneine (EGT) is a high-value antioxidant for food, pharmaceutical, nutraceutical, and cosmetic applications. Microbial production from renewable feedstocks is promising, but efficient biosynthesis requires coordinated precursor supply. Precursor-supplementation experiments indicated that multiple amino acids may jointly influence EGT production. Based on pathway biochemistry and previous engineering evidence, endogenous l-cysteine supply was selected as one mechanistically relevant engineering target. Here, an Escherichia coli platform for glucose-derived EGT production was developed by combining biosensor-driven evolution and transcriptome-guided chassis engineering. A dual-output l-cysteine-responsive biosensor linked kanamycin resistance and fluorescence, enabling sequential growth-based enrichment and fluorescence-based prioritization. Mutagenesis, microdroplet-assisted adaptive evolution, and single-cell sorting generated an evolved chassis with 47.10% higher extracellular l-cysteine accumulation and 64.49% higher EGT production. Transcriptome-guided bsmA activation further improved production, and the final strain produced 1.20 g/L EGT in a 3 L fed-batch fermentation without additional l-cysteine supplementation.