Complete integration of carbene-transfer chemistry into biosynthesis

Biosynthesis is an environmentally friendly and renewable way to synthesize a broad range of natural and some new-to-nature products; however, it lacks many of the reactions and flexibility of synthetic chemistry, an example being carbene mediated reactions. While it was recently shown that these reactions can be imported into the cell, carbene donors and unnatural cofactors needed to be added exogenously to the cells, precluding cost-effective scale-up of the reaction. Here we identified a biosynthetic gene cluster that when expressed in Streptomyces albus will produce the diazo compound azaserine, which we also demonstrated can serve as a carbene donor across a carbon-carbon double bond in another intracellularly produced molecule, styrene, thereby producing a cyclopropanated product. The reaction was catalyzed with P450 mutants harboring a native cofactor with excellent diastereoselectivity and moderate yield. This work establishes a platform for introducing carbene mediated reactions into microbes for bio-manufacture and contributes to sustainable green chemistry.