TY - JOUR
T1 - Preparation, characterization, and optimization of an in vitro C 30 carotenoid pathway
AU - Ku, Bosung
AU - Jeong, Jae Cheol
AU - Mijts, Benjamin N.
AU - Schmidt-Dannert, Claudia
AU - Dordick, Jonathan S.
PY - 2005/11
Y1 - 2005/11
N2 - 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.
AB - 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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U2 - 10.1128/AEM.71.11.6578-6583.2005
DO - 10.1128/AEM.71.11.6578-6583.2005
M3 - Article
C2 - 16269684
AN - SCOPUS:32044450527
SN - 0099-2240
VL - 71
SP - 6578
EP - 6583
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
IS - 11
ER -