Cascade biocatalysis for pyridoxal 5′-phosphate synthesis with ATP autonomy via polyphosphate kinase

The industrial production of pyridoxal 5′-phosphate (PLP)—an indispensable enzymatic cofactor—is hampered by inefficient multistep chemical syntheses and the economic infeasibility of ATP-dependent biocatalytic routes. To address these challenges, we report an ATP-autonomous biocatalytic cascade that couples pyridoxal kinase (PLK) with polyphosphate kinase (PPK) for sustainable PLP synthesis. This system harnesses inexpensive sodium hexametaphosphate (SHMP) as a phosphoryl donor to drive continuous ATP regeneration, thereby enabling efficient phosphorylation of pyridoxal to PLP without exogenous ATP input. Recombinant PLK and PPK were co-expressed in E. coli BL21(DE3), yielding specific activities of 50 and 145 U/g cell , respectively. The whole-cell biocatalyst achieved only about 25% conversion yield due to mass transfer limitations and intracellular PLP degradation. Notably, while the optimized cell-free system (45 °C, pH 6.5, PLK:PPK = 1:3, 25 mM SHMP) attained a remarkable 95% PLP yield. This 3.8-fold enhancement demonstrates the critical advantage of eliminating cellular barriers in complex multi-enzyme cascades. Our work establishes a robust, ATP-independent platform for high-efficiency PLP biosynthesis that seamlessly integrates cofactor regeneration with cascade catalysis, offering a scalable and economically viable route for industrial biomanufacturing. Moreover, it demonstrates the broader utility of polyphosphate-driven energy recycling in sustainable biocatalysis.