High-level de novo biosynthesis of pyridoxal 5'-phosphate in Bacillus licheniformis by systems engineering.

Abstract

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, is an essential cofactor with broad applications in the pharmaceutical and food industries. However, its microbial production is hindered by intracellular toxicity, tight metabolic regulation, degradation, and limited export. This study aimed to develop an efficient de novo PLP production platform in Bacillus licheniformis by integrating metabolic, transporter, and enzyme engineering strategies. We first assessed PLP tolerance and found that B. licheniformis tolerated exogenous PLP exposure, suggesting its potential as a production host. A xylose-inducible system was then introduced to dynamically control PLP synthase expression, thereby minimizing the adverse effects of early PLP accumulation. Subsequently, PLP biosynthesis was enhanced by improving the supply of ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine through modular pathway optimization. In parallel, PLP degradation was reduced and a heterologous transporter was introduced to promote extracellular PLP accumulation. Furthermore, protein engineering of the PLP synthase complex was performed to improve its catalytic efficiency. The resulting strain produced 1.5 g/L PLP with a productivity of 20.8 mg/L/h in a 5-L bioreactor, the highest titer reported for microbial de novo PLP production. This work offers new insight into boosting microbial PLP production and holds considerable potential for industrial applications.

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