A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3)

TitleA novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3)
Publication TypeJournal Article
Year of Publication2009
AuthorsDietrich, J. A., Yoshikuni Y., Fisher K. J., Woolard F. X., Ockey D., McPhee D. J., Renninger N. S., Chang M. C. Y., Baker D., & Keasling J. D.
JournalACS chemical biology
Volume4
Issue4
Pagination261-7
Date Published2009 Apr 17
ISSN1554-8937
KeywordsAlgorithms, Artemisinins, Bacillus megaterium, Bacterial Proteins, Catalytic Domain, Collaborative Publication, Computer Simulation, Crystallography, X-Ray, Cytochrome P-450 Enzyme System, Models, Molecular, Molecular Conformation, Mutation, NADPH-Ferrihemoprotein Reductase, Oxidation-Reduction, Protein Engineering, Sesquiterpenes, Stereoisomerism, Time Factors
Abstract

Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.

Alternate JournalACS Chem. Biol.
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