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  • Title: Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway.
    Author: Ku B, Jeong JC, Mijts BN, Schmidt-Dannert C, Dordick JS.
    Journal: Appl Environ Microbiol; 2005 Nov; 71(11):6578-83. PubMed ID: 16269684.
    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.
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