Specific transformation of steviol at unactivated C-H sites by microorganism and mining of key P450 enzymes

Enzyme engineering has witnessed substantial advancements, particularly in the application of intricate microbial enzyme systems to substrate modification. Steviol (ST), a diterpenoid compound characterised by a typical shell diene skeleton, was subjected to biotransformation using 50 species of microorganisms. Among these, Fusarium graminearum PH-1 strain effectively converted ST to a novel derivative, 2-β-hydroxy-steviol (2-β-OH-ST, STH). Subsequent treatment with piperonyl butoxide (PBO) or Fe2+ inferred the involvement of cytochrome P450 enzymes in this conversion. A crucial CYP450 gene, P450-10, was identified via yeast overexpression and exhibited an in vitro catalytic efficiency of 18.7%. Orthomutation investigations further revealed that L94A, F98A, and M281A mutants entirely lost catalytic activity. Lastly, STH demonstrated markedly enhanced anti-inflammatory activity compared to ST. Moreover, its esterified derivative, 19- methyl −2- hydroxysteviol (STHE), markedly inhibited the growth of PC9 cells with IC50 value of 416.8 μmol/L. The research presents an innovative strategy for the regioselective inactive C-H sites in ST and provides a fresh structural foundation for the development of potential therapeutic agents.