Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway

Appl Environ Microbiol. 2005 Nov;71(11):6578-83. doi: 10.1128/AEM.71.11.6578-6583.2005.


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

  • Evaluation Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biotechnology / methods
  • Carotenoids / chemistry
  • Carotenoids / metabolism*
  • Cloning, Molecular
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Farnesyl-Diphosphate Farnesyltransferase / genetics
  • Farnesyl-Diphosphate Farnesyltransferase / metabolism*
  • Geranyltranstransferase / genetics
  • Geranyltranstransferase / metabolism*
  • Hemiterpenes / metabolism
  • Kinetics
  • Organophosphorus Compounds / metabolism
  • Polyisoprenyl Phosphates / metabolism
  • Sesquiterpenes


  • Bacterial Proteins
  • Escherichia coli Proteins
  • Hemiterpenes
  • Organophosphorus Compounds
  • Polyisoprenyl Phosphates
  • Sesquiterpenes
  • isopentenyl pyrophosphate
  • 3,3-dimethylallyl pyrophosphate
  • Carotenoids
  • farnesyl pyrophosphate
  • CrtM protein, Staphylococcus aureus
  • Geranyltranstransferase
  • Farnesyl-Diphosphate Farnesyltransferase