Rational Design of Salmonella typhi Acid Phosphatase for Efficient Production of Pyridoxal 5'-Phosphate.

Pyridoxal 5'-phosphate (PLP) is highly valuable in food and medicine. However, achieving the efficient biosynthesis of PLP remains challenging. Here, a salvage pathway using acid phosphatase from Salmonella typhi ( St APase) and pyridoxine oxidase from Escherichia coli ( Ec PNPO) as pathway enzymes was established for the first time to synthesize PLP from pyridoxine (PN) and pyrophosphate (PPi). St APase was identified as a rate-limiting enzyme. Two protein modification strategies were developed based on the PN phosphorylation mechanism: (1) improving the binding of PN into St APase and (2) enhancing the hydrophobicity of St APase's substrate binding pocket. The k cat / K m of optimal mutant M7 was 4.9 times higher than that of the wild type. The detailed mechanism of performance improvement was analyzed. Under the catalysis of M7 and Ec PNPO, a PLP high-yielding strain of 14.5 ± 0.55 g/L was engineered with a productivity of 1.0 ± 0.02 g/(L h) (the highest to date). The study suggests a promising method for industrial-scale PLP production.