Abstract
A simple, rapid, and inexpensive extraction method for carotenoids and other non-polar compounds present in phototrophic bacteria has been developed. The method, which has been extensively tested on the phototrophic purple non-sulphur bacterium Rhodospirillum rubrum, is suitable for extracting large numbers of samples, which is common in systems biology studies, and yields material suitable for subsequent analysis using HPLC and mass spectroscopy. The procedure is particularly suitable for carotenoids and other terpenoids, including quinones, bacteriochlorophyll a and bacteriopheophytin a, and is also useful for the analysis of polar phospholipids. The extraction procedure requires only a single step extraction with a hexane/methanol/water mixture, followed by HPLC using a Spherisorb C18 column, with a mobile phase consisting of acetone-water and a non-linear gradient of 50%–100% acetone. The method was employed for examining the carotenoid composition observed during microaerophilic growth of R. rubrum strains, and was able to determine 18 carotenoids, 4 isoprenoid-quinones, bacteriochlorophyll a and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The analytical procedure was used to examine the dynamics of carotenoid biosynthesis in the major and minor pathways operating simultaneously in a carotenoid biosynthesis mutant of R. rubrum.
Highlights
IntroductionRapid methods for chemical analysis of metabolic intermediates are becoming increasingly important in many areas, in particular for systems biology studies of the cellular metabolome
Rapid methods for chemical analysis of metabolic intermediates are becoming increasingly important in many areas, in particular for systems biology studies of the cellular metabolome.An extremely active area here is the optimization of terpenoid metabolism in bacteria, where pathway design principles are employed to achieve overproduction of industrially interesting carotenoids and quinones
We have recently demonstrated for the first time that the phototrophic bacterium, Rhodospirillum rubrum, which normally produces the purple carotenoid spirilloxanthin almost exclusively, can be genetically modified to overproduce the carotenoid lycopene, a carotenoid that normally only occurs at very low amounts in this organism [8]
Summary
Rapid methods for chemical analysis of metabolic intermediates are becoming increasingly important in many areas, in particular for systems biology studies of the cellular metabolome. An extremely active area here is the optimization of terpenoid metabolism in bacteria, where pathway design principles are employed to achieve overproduction of industrially interesting carotenoids and quinones. This area is confounded by the necessity to perform organic extractions of cellular material, for which methods are well established in the literature. General methods for the extraction of carotenoids and quinones which are compatible with HPLC-MS analysis have been presented [1,2]. These methods have been shown to be highly efficient and can be used for the extraction of terpenoids from a wide variety of cellular sources
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
