Modular Chemistries in E. coli for Biomanufacturing of Natural Products

The recreation of natural product biosynthetic pathways from eukaryotes for sustainable commercial production in prokaryotic hosts is fraught with challenges, such as inefficient enzymes, poor protein solubility and misfolding, and the absence of organelles. Biomanufacturing of natural products requires the controlled interplay of multiple complex chemistries in a single cell – and in a prokaryote, a single chemical environment. Oxygenation is one of the most challenging chemical modifications required for the synthesis of natural products. Despite being widely used in microbial fermentation, E. coli has been described as an unsuitable host for recombinant pathways that include the eukaryotic cytochrome P450s required for synthesis of many natural products. We demonstrate that oxygenated terpenoids can be selectively produced by E. coli incorporating one or two plant CYP450s. These CYP450s have been engineered for novel or enhanced activity, solubility, and stability to enable high-titer production in E. coli cultures. We further integrate in vivo glycosylation chemistry into the strains, greatly expanding the functional chemical space exploitable by E. coli. Our results are demonstrated with examples including Taxol and rebaudioside M production, representative of the diverse applications made possible by this platform.