Preparation, characterization, and optimization of an in vitro C 30 carotenoid pathway

Bosung Ku, Jae Cheol Jeong, Benjamin N. Mijts, Claudia Schmidt-Dannert, Jonathan S. Dordick

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)6578-6583
Number of pages6
JournalApplied and environmental microbiology
Volume71
Issue number11
DOIs
StatePublished - Nov 2005

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