Combination of adaptive laboratory evolution and metabolic engineering on Vibrio natriegens for efficient production...

Ethanol is a non-food carbon source that is derived from syngas fermentation and has the potential to serve as a raw material for microbial processes that produce various value-added chemicals. Vibrio natriegens is a new type of chassis cell that has been developed in recent years for use in biotechnology and synthetic biology. It has a very fast growth rate and a broad substrate spectrum, but its ability to utilize ethanol is limited and its tolerance is poor. Adaptive Laboratory Evolution (ALE) provides a powerful tool for studying the resistance phenotype, production activity, and genetic stability of industrial strains, and is widely used to screen engineered strains with further improved characteristics. In this study, we developed a Vibrio natriegens that evolved in ethanol to produce 3-hydroxypropionic acid (3-HP). Firstly, strains capable of producing 3-HP from ethanol were obtained through adaptive laboratory evolutionary screening. Subsequently, by optimization of promoters in the 3-HP biosynthetic pathways, rational design of background strain, and adding appropriate concentrations of cerulenin, the 3-HP biosynthesis of engineered strain was significantly improved. Finally, the maximum concentration of 3-HP in shake flask culture reached 3.10 g/L, with a yield of 0.382 g/g. The concentration of 3-HP in 1 L bioreactor fermentation reached 9.94 g/L, achieving a yield of 0.258 g/g. The findings suggest that ethanol derived from synthesis gas shows tremendous promise as an excellent carbon feedstock for producing high-value biochemicals using engineered Vibrio natriegens in integrated carbon-based industrial biotechnology.