A Two-Enzyme Entry Module Triggers an Endogenous Biocatalytic Cascade for Green Biosynthesis of Pyridoxal 5′-Phosphate in Corynebacterium glutamicum

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor, yet its industrial supply still relies largely on multi-step chemical synthesis. Here, using the industrial chassis Corynebacterium glutamicum ATCC 13032, we proposed and validated a strategy based on a minimal heterologous entry coupled to endogenous pathway continuation, resulting in a distinct PLP-producing route. Three engineered strains were constructed and compared: S1 expressing ecepd from Escherichia coli; S2 co-expressing ecepd plus ecpdxB from Escherichia coli (a minimal two-gene module); and S3 carrying an additional ecpdxA from Escherichia coli and smpdxJ from Sinorhizobium meliloti to form a four-gene module as a benchmark for heterologous reconstruction. The wild-type (WT) strain produced a basal PLP level of 10.6 mg/L. Overexpressing ecepd alone increased the titer to 40.4 mg/L (3.8-fold vs WT), whereas the minimal two-gene module in S2 yielded the highest PLP titer of 95.5 mg/L (9.0-fold vs WT; 136.0% higher than S1). Notably, the four-gene module (S3) reached 70.0 mg/L, which was 36.3% lower than S2 under matched conditions. These results indicated that introducing only a minimal two-gene entry could cooperate with the endogenous metabolic network of Corynebacterium glutamicum to establish a new and highly effective PLP biosynthetic route, with production performance exceeding that of a multi-gene heterologous reconstruction in the tested window. This work provides a low-burden and scalable framework for sustainable PLP biomanufacturing and motivates further optimization targeting the endogenous continuation steps and regulatory constraints.