Synthesis of flavonoid phosphate derivatives via a coupled biocatalytic system involving ATP regeneration

Abstract: ATP-dependent enzymes are involved in biosynthesis of diverse chemicals. We previously identified a novel phosphotransferase from Bacillus subtilis, namely BsFPS, which enables phosphorylation of flavonoids to generate water-soluble monophosphate derivatives, highlighting the potential of BsFPS as a bioconversion platform to solve solubility issues for nutraceutical applications. Nevertheless, as an ATP-dependent enzyme, adding stoichiometric quantities of expensive ATP poses challenges to the biocatalytic processes for industrial implementation. Here, we reported a coupled enzyme system for producing flavonoid phosphates, which consists of BsFPS and a class III polyphosphate kinase 2 (PPK2-III) for ATP regeneration from AMP and a cheap phosphate donor, polyphosphate, thus reducing ATP consumption. Based on the known biochemical properties of BsFPS (40℃, pH 7.5, Mg²⁺/Mn²⁺), we first selected PPK12, a PPK2-III from biogas fermenter metagenome, as candidate since it was reported to generate ATP under a compatible condition with BsFPS. We revealed PPK12 displayed a higher specific activity at 40℃ and pH 7.5 than that of BsFPS using Mg²⁺ as cofactor, suggesting the feasibility to involve ATP regeneration in the reaction system. In addition, we found PPK12 preferred to use sodium hexametaphosphate (polyP₆) rather than short chain polyphosphates (i.e. sodium triphosphate and sodium trimetaphosphate). By combining BsFPS and PPK12 to establish the coupled enzyme system, a final yield of 6.68 ± 0.58 mM luteolin phosphates from 9 mM luteolin, 1 mM ATP, and 20 mM polyP₆ was achieved. This study provides the first enzymatic route for synthesis of luteolin phosphates by coupling BsFPS and PPK12 to enable ATP recycling in vitro, which reduces up to 90% ATP usage, paving the way for further scale-up production of novel flavonoid phosphates. Our findings broaden the potential of oilseeds and herbs which are natural sources of flavonoids to develop new functional uses.