(164aj) Introduction of Abiological Reactions into Biosynthesis

Reactions in chemistry are much more abundant and diverse than in biology. If those reactions in chemistry can be brought into microorganisms, it will broaden the types of compounds that can be biosynthesized inside the cell and offer the possibility for diversifying valuable natural compounds in vivo, such as medicine precursors. Carbene-mediated reactions have wide applications in chemical synthesis, but occur rarely in biology. Here we report the introduction of a carbene-mediated reaction with a biosynthesized carbene precursor into a microbe and integration of it with a natural biosynthesis pathway to generate new compounds. The biosynthesis pathway for the carbene precursor was identified, cloned and heterologously expressed to produce the precursor. Natural P450s were screened and improved by directed evolution to catalyze the reaction between the biosynthesized carbene precursor and the other substrate, which is also biosynthesized by introducing the corresponding natural pathway, with moderate yield and excellent diastereoselectivity. In this case, all the components involved in the abiological reaction, the carbene precursor, the other substrate and the enzyme catalyzing the reaction, are biosynthesized. This work sets a platform for introducing more abiological carbene mediated or related reactions into microbes for bio-manufacturing and contributes to green chemistry.