Two-step bioconversion of pyridoxal 5′-phosphate from pyridoxine through cofactor regeneration and pyridoxine 5′-phosphate oxidase evolution

compared to the wild-type enzyme. Using a two-step bioconversion process, we achieved the production of 35.2 g/L pyridoxine 5'-phosphate (PNP) with a molar conversion of 94.7%, and 21.3 g/L PLP, corresponding to a final molar conversion yield of approximately 57.5% from PN in a 1-L bioreactor. This work demonstrates an efficient, sustainable and scalable strategy for the industrial production of PLP.