Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway.
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.
Ku
Bosung
B
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Jeong
Jae-Cheol
JC
Mijts
Benjamin N
BN
Schmidt-Dannert
Claudia
C
Dordick
Jonathan S
JS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000378:metabolism*; D051228:Geranyltranstransferase / Q000235:genetics / Q000378:metabolism*; D045782:Hemiterpenes / Q000378:metabolism; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012717:Sesquiterpenes
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D002338:Carotenoids; C004808:farnesyl pyrophosphate; C091056:CrtM protein, Staphylococcus aureus; D051228:Geranyltranstransferase; D013186:Farnesyl-Diphosphate Farnesyltransferase