Metabolic engineering of Vibrio natriegens for the efficient biosynthesis of ergothioneine from sucrose using...

Fast-growing Vibrio natriegens is now recognized as a next-generation chassis for synthetic biology and biotechnology; however, its low transformation efficiency, limited gene editing methods and high fermentation cost are still the main challenges hampering its industrial application. In this study, we established an efficient electroporation transformation and dual-plasmid CRISPR-Cas9 editing system in V. natriegens. Subsequently, the heterologous ergothioneine biosynthetic pathway involving the combination of the superstrong P_L lacO1 promoter and weak RBS7 was constructed in V. natriegens. Multiple genes encoding genes involved in byproduct formation and adenosine triphosphate (ATP) degradation were consecutively deleted, while several key genes involved in the S-adenosylmethionine (SAM) cycle and the ATP synthesis pathway were overexpressed to increase ergothioneine production. Finally, fed-batch fermentation was performed using low-cost sucrose as the sole carbon source under high-salinity, non-sterile conditions, resulting in an ergothioneine titer of 1.2 g/L in a 2-L bioreactor. This study not only provides the first successful example of the ergothioneine biosynthesis with engineered V. natriegens strains but also establishes an efficient and economic platform in which V. natriegens is used to produce other high-value compounds.