细胞色素P450的蛋白质工程使这些生物催化剂能够促进自然界以外的各种非生物反应。将此类非天然转化与微生物生物合成途径整合可以允许修饰的天然产物衍生物的可持续酶促生产。特别是,三氟甲基化是药物研究中非常理想的修饰,由于三氟甲基对药物效力的积极影响,生物利用度,和代谢稳定性。这项研究证明了使用工程细胞色素P450变体的天然单萜支架的非天然三氟甲基取代的环丙烷衍生物的生物合成,P411-PFA.P411-PFA成功催化了三氟甲基卡宾从2-重氮-1,1,1-三氟乙烷转移到几种单萜的末端烯烃,包括L-carveol,Carvone,紫苏酒精,还有Perillartine,生成相应的三氟甲基化环丙烷产品。此外,将这种非生物环丙烷化反应与大肠杆菌中L-carveol生产的重建代谢途径相结合,可以一步生物合成柠檬烯前体的三氟甲基化L-carveol衍生物。总的来说,将合成酶化学与已建立的代谢途径合并代表了可持续生产生物活性天然产物类似物的有希望的方法。
Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature\'s repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic
cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.