Abstract
The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4'-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.
Publication types
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Evaluation Study
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism*
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Biotechnology / methods
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Carotenoids / chemistry
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Carotenoids / metabolism*
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Cloning, Molecular
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Escherichia coli / enzymology*
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Escherichia coli / genetics*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Farnesyl-Diphosphate Farnesyltransferase / genetics
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Farnesyl-Diphosphate Farnesyltransferase / metabolism*
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Geranyltranstransferase / genetics
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Geranyltranstransferase / metabolism*
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Hemiterpenes / metabolism
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Kinetics
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Organophosphorus Compounds / metabolism
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Polyisoprenyl Phosphates / metabolism
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Sesquiterpenes
Substances
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Bacterial Proteins
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Escherichia coli Proteins
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Hemiterpenes
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Organophosphorus Compounds
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Polyisoprenyl Phosphates
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Sesquiterpenes
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isopentenyl pyrophosphate
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3,3-dimethylallyl pyrophosphate
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Carotenoids
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farnesyl pyrophosphate
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CrtM protein, Staphylococcus aureus
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Geranyltranstransferase
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Farnesyl-Diphosphate Farnesyltransferase