Protein engineering and iterative multimodule optimization for vitamin B6 production in Escherichia coli

Vitamin B₆ is an essential nutrient with extensive applications in the medicine, food, animal feed, and cosmetics industries. Pyridoxine (PN), the most common commercial form of vitamin B₆, is currently chemically synthesized using expensive and toxic chemicals. However, the low catalytic efficiencies of natural enzymes and the tight regulation of the metabolic pathway have hindered PN production by the microbial fermentation process. Here, we report an engineered Escherichia coli strain for PN production. Parallel pathway engineering is performed to decouple PN production and cell growth. Further, protein engineering is rationally designed including the inefficient enzymes PdxA, PdxJ, and the initial enzymes Epd and Dxs. By the iterative multimodule optimization strategy, the final strain produces 1.4 g/L of PN with productivity of 29.16 mg/L/h by fed-batch fermentation. The strategies reported here will be useful for developing microbial strains for the production of vitamins and other bioproducts having inherently low metabolic fluxes.