A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3).
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.
Dietrich
Jeffrey A
JA
UCSF/UCB Joint Graduate Group in Bioengineering, University of California at Berkeley, Berkeley, CA 94720, USA.
Yoshikuni
Yasuo
Y
Fisher
Karl J
KJ
Woolard
Frank X
FX
Ockey
Denise
D
McPhee
Derek J
DJ
Renninger
Neil S
NS
Chang
Michelle C Y
MC
Baker
David
D
Keasling
Jay D
JD
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
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