Abstract

In recent years, the direct synthesis of l-cysteine from l-serine and sodium hydrosulfide catalyzed by tryptophan synthase (TrpS) has attracted considerable attention. However, under high substrate concentration conditions, the conversion efficiency of TrpS remains limited, which restricts its industrial application. To enhance catalytic efficiency, this study developed a directed evolution platform integrating in vivo continuous mutagenesis, a highly sensitive l-cysteine biosensor, and fluorescence-activated cell sorting for high-throughput screening. Using this platform, multiple TrpS variants were successfully obtained, among which the combinatorial mutant V139M/A302P exhibited superior catalytic activity and stability compared with the wild type. Under optimized reaction conditions, this mutant achieved an l-cysteine titer of 116.4 g/L. Furthermore, by introducing a whole-cell immobilization strategy, the immobilized cells produced a maximum l-cysteine titer of 113.05 g/L in a single batch, with a conversion rate of 93.3%. This study establishes an efficient, stable, and industrially promising platform for l-cysteine biosynthesis and screening.