Biocatalytic Production of Pyridoxal 5′-Phosphate: Enzyme Engineering, Phosphate-Donor Economy, and Process-Readiness of Salvage Cascades

Pyridoxal 5′-phosphate (PLP), the catalytically active form of vitamin B6, is an enabling cofactor for synthetic biocatalysis, but PLP production remains difficult to compare across chemical, microbial, and cell-free routes. This review reframes PLP synthesis as a biocatalytic cascade-design problem. Chemical phosphorylation is used as a benchmark for selectivity, reagent burden, and purification, whereas de novo and salvage-pathway enzymes define the molecular constraints governing biological production. We focus on PdxK/PdxY, PdxH/PNPOx, PdxS/PdxT, engineered acid phosphatase, and polyphosphate kinase modules. Recent PPi-driven and PPK/polyP-supported cascades show that high PLP concentrations are attainable, but titer alone is not sufficient for route comparison: substrate identity, phosphate-donor equivalents, adenylate loading, whole-cell or cell-free format, oxygen and H2O2 management, catalyst reuse, salt burden, isolated recovery, and product purity must be evaluated separately. By distinguishing PN fermentation, intracellular cofactor supply, PNP intermediates, reaction-broth PLP concentration, and isolated PLP yield, this review proposes a route-readiness map for PLP-producing cascades. The most promising next-generation systems will couple product-tolerant phosphorylation, oxidase performance, cofactor regeneration, and downstream stabilization in a single process-aware design.