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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Algorithms
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Artemisinins / chemistry
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Artemisinins / metabolism*
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Bacillus megaterium / enzymology*
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism*
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Catalytic Domain
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Computer Simulation
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Crystallography, X-Ray
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Cytochrome P-450 Enzyme System / chemistry
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism*
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Models, Molecular
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Molecular Conformation
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Mutation
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NADPH-Ferrihemoprotein Reductase / chemistry
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NADPH-Ferrihemoprotein Reductase / genetics
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NADPH-Ferrihemoprotein Reductase / metabolism*
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Oxidation-Reduction
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Polycyclic Sesquiterpenes
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Protein Engineering*
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Sesquiterpenes / chemistry
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Sesquiterpenes / metabolism
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Stereoisomerism
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Time Factors
Substances
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Artemisinins
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Bacterial Proteins
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Polycyclic Sesquiterpenes
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Sesquiterpenes
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amorpha-4,11-diene
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dihydroartemisinic acid
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Cytochrome P-450 Enzyme System
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NADPH-Ferrihemoprotein Reductase
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flavocytochrome P450 BM3 monoxygenases