Biocatalytic cascade for the synthesis of methylphosphonate from phosphoenolpyruvate

Methylphosphonate (Mpn), a building block for complex organophosphonate, is utilized in pharmaceuticals, agriculture, and chemical industries. It also serves as a critical substrate for resolving the methane paradox in ecological studies. However, current Mpn synthesis predominantly relies on chemical methods. Therefore, there is a growing interest in developing biosynthetic approaches for Mpn production. In this study, the biosynthetic pathway of Mpn was reconstituted. Four crucial enzymes involved in the conversion of phosphoenolpyruvate (PEP) to Mpn were screened: phosphoenolpyruvate mutase (AepX), phosphonopyruvate decarboxylase (AepY), phosphonoacetaldehyde reductase (AlpJ), and methylphosphonate synthase (MpnS). Both in vitro and in vivo biocatalytic cascades were implemented. Through systematic optimization of the in vitro reaction conditions, a final Mpn conversion yield of 76% was achieved from 5 mM PEP, with an optimal enzyme concentration ratio of 5 µM AepX, 10 µM AepY, 10 µM AlpJ, and 10 µM MpnS. Building on the in vitro system, recombinant Escherichia coli strains co-expressing four enzymes were engineered as whole-cell catalysts. By employing a dual-plasmid system with varying copy numbers to regulate heterologous enzyme expression, engineered strains with distinct synthetic capabilities were obtained. The engineered strain E6 (harboring plasmids pCDFDuet-aepX-aepY and pETDuet-alpJ-mpnS) produced 7.19 mM Mpn, corresponding to a 35.95% molar conversion yield within 16 h. This study established a biosynthetic method for Mpn production from PEP through enzymatic cascade, operating under mild aqueous conditions.