A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat Chem Biol 9

Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a unique serine-heme ligated cytochrome “P411 ” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450BM3 gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state FeIII/II reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity. Genetically programmed whole-cell biocatalysts are readily produced in simple growth media, do not require further purification or isolation and can be engineered with biosynthetic pathways for the elaboration of complex molecules1–3. The range of accessible transformations, however, is currently limited to the chemical repertoire of natural enzymes. Designing enzymes for non-natural reactions in vivo has been challenging due to the requirements for assembly of the functional catalyst, the compatibility of synthetic reagents in the cellular milieu, and cell permeability to allow substrate influx and product release. The catalysis of non-natural transformations inside cells will enable alternative biosynthetic routes to natural and artificial products, biocatalytic production of chemicals currently made using synthetic reactions, and will expand the chemical toolbox available for in vivo studies of cellular function4. We recently showed that a few amino acid mutations in a bacterial cytochrome P450 monooxygenase can unlock significant cyclopropanation activity in vitro. Variants of P450BM3 from Bacillus megaterium catalyze hundreds of turnovers of formal carben