title: A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3).
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.
authors.affiliation: UCSF/UCB Joint Graduate Group in Bioengineering, University of California at Berkeley, Berkeley, CA 94720, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Dietrich
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshikuni
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Fisher
authors.forename: Frank X
authors.initials: FX
authors.lastname: Woolard
authors.forename: Denise
authors.initials: D
authors.lastname: Ockey
authors.forename: Derek J
authors.initials: DJ
authors.lastname: McPhee
authors.forename: Neil S
authors.initials: NS
authors.lastname: Renninger
authors.forename: Michelle C Y
authors.initials: MC
authors.lastname: Chang
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000465:Algorithms; D037621:Artemisinins / Q000737:chemistry / Q000378:metabolism*; D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D000081223:Polycyclic Sesquiterpenes; D015202:Protein Engineering; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D037621:Artemisinins; D001426:Bacterial Proteins; D000081223:Polycyclic Sesquiterpenes; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; C520462:dihydroartemisinic acid; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases