Exploring P450 Expression in Escherichia coli for the Synthesis of Complex Molecules

Biocatalysis projects to play a significant role in the future of chemical synthesis, especially for the creation of complex natural products, their analogs, and new chemical entities built upon their scaffolds. This is due to the ability of enzymes to leverage the incredible precision of a three dimensional binding pocket to perform highly specific functionalizations, overcoming a molecule’s inherent reactivity biases. Added within the context of metabolic engineering, enzymatic biocatalysis possesses tremendous opportunity to improve scalability, minimize environmental impact, and reduce costs by employing fast growing organisms, performing reactions at near neutral conditions, and converting affordable feedstock to compounds of interest.

Of the potential chemical targets, terpenoids, the largest class of plant specialized metabolites, are particularly interesting as they hold relevance to flavor, fragrance, pharmaceutical, and fuel targets. Realizing the full impact of terpenoids, though, will hinge upon our ability to efficiently perform complex functionalizations of terpene backbone molecules. To this end, harnessing the capabilities of the P450 family of enzymes will be essential, as they represent nature’s oxygenase of choice for functionalizing unreactive carbons with specificity. Herein we present our work aimed at obtaining high titers of functionalized terpenoid molecules in the Taxol biochemical pathway. We will focus on functional expression of plant P450s in E. coli, balancing metabolic pathway flux, improving catalytic efficiency by modulating reductase partners (CPR), and performing detailed characterization of P450 enzymes with in vitro assays, all in order to construct high-performing systems.