Combinatorial Metabolic Engineering of <i>Escherichia coli</i> MG1655 Enables Efficient Biosynthesis of Vitamin B₆

Pyridoxine (PN) is the most important commercial form of vitamin B₆. Currently, PN is chemically synthesized using toxic chemicals, causing environmental pollution. Microbial production of vitamin B₆ offers a more sustainable alternative, but its low productivity makes it difficult to meet commercialization demands. Here, metabolic engineering strategies were used to efficiently improve the PN production. Directed evolution of 4-phosphoerythronate dehydrogenase led to a 4-fold increase in PN production. In addition, the <i>pdxK</i> and <i>pdxH</i> genes were then knocked out to redirect the metabolic flux from pyridoxine 5'-phosphate to PN, and genes involved in glycerol-to-pentose phosphate metabolism were overexpressed to increase precursors for PN synthesis. Finally, the recombinant strain produced 2120.1 ± 7.8 mg/L PN with a productivity of 32.1 mg/L/h by batch fermentation. The strategies reported here will be useful for the efficient and sustainable biobased production of PN.