Coupling a rebuild shuttle system with biosynthetic pathway and transcription factor engineering for enhanced l-cysteine production†

l-Cysteine, a vital sulfur-containing amino acid, is extensively utilized in the pharmaceutical, food, cosmetics, and feed industries; it also plays a crucial role in the sulfur cycle on the Earth. Here, we report the development of a plasmid-free engineered Escherichia coli strain for enhanced l-cysteine production. Initially, the l-cysteine/l-cystine shuttle system was restructured to adapt to the new engineered strain. For the critical metabolic node CysE, a genomic multi-copy strategy was employed to enhance its expression extensively, thereby increasing the metabolic flux of the carbon module. A substrate channel for l-cysteine was then constructed to enhance biosynthetic efficiency. The multi-module co-localization strategy was designed to couple the module enzyme with the efflux system, which coordinates the biosynthesis and transport of the product. Moreover, a CysB mutant was screened to promote sulfur assimilation globally. By enhancing the carbon and sulfur module, the strain GCB2 produced 35.54 g L⁻¹ of l-cysteine in a 5 L bioreactor, with a glucose yield of 0.125 g g⁻¹, a sulfur assimilation of 92.44%, and a productivity of 0.555 g L⁻¹ h⁻¹. To our knowledge, this is the highest-known production, laying the foundation for future industrial applications. The strategy we developed in this study can also be applied for the production of other chemicals.