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Applied and Environmental Microbiology, November 2005, p. 6578-6583, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6578-6583.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Preparation, Characterization, and Optimization of an In Vitro C30 Carotenoid Pathway

Bosung Ku,1 Jae-Cheol Jeong,1 Benjamin N. Mijts,2 Claudia Schmidt-Dannert,2 and Jonathan S. Dordick1*

Department of Chemical and Biological Engineering, Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180,1 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 551082

Received 9 May 2005/ Accepted 27 June 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.

* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180. Phone: (518) 276-2899. Fax: (518) 276-2207. E-mail: dordick{at}rpi.edu.

Applied and Environmental Microbiology, November 2005, p. 6578-6583, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6578-6583.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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