Unnatural Biosynthesis by an Engineered Microorganism with Heterologously Expressed Natural Enzymes and an Artificial Metalloenzyme

<title>Abstract</title> Synthetic biology enables microbial hosts to produce complex molecules that are otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by chemical reactions catalyzed by natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyze unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically by microorganisms. We report an engineered microbial cell expressing a heterologous biosynthetic pathway, which contains both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. To create this hybrid biosynthetic organism, we engineered Escherichia coli (E. coli) with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titer of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, together with natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.