title: Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry.
abstract: A method employing silicone oil density centrifugation, solid-phase extraction (SPE) cleanup, and LC-ESI-MS/MS analysis was developed for the rapid, selective, sensitive, and quantitative detection of an intracellular pool of short organic acid-CoA esters in actinomycetes. The detection limit was determined to be approximately 0.8 pmol (1.2 ng/ml) for each standard CoA-ester analyzed by the present LC-ESI-MS/MS method. A selected ion chromatogram for a typical fragment ion (m/z 428) specific to CoA-esters enabled the detection of eight intracellular CoA-esters involved in both primary and secondary metabolisms. The application of this method to bacterial metabolomic study is demonstrated by the profiling of the intracellular CoA-ester pools in the wild-type Streptomyces venezuelae strain producing polyketide antibiotics (methymycin and pikromycin), a polyketide synthase (PKS)-deleted S. venezuelae mutant, and a S. venezuelae mutant expressing the heterologous PKS genes. By quantifying the individual CoA-esterlevel in three different genotypes of the S. venezuela e strain, further insight could be gained into the role of CoA-estersin polyketide biosynthesis. This analytical approach can be extended to the quantification of the size and composition of in vivo CoA-ester pools in various microbes, and can provide a detailed understanding of the relationship between the in vivo CoA-ester pool and the production of pharmaceutically important polyketides.
authors.affiliation: Division of Nano Sciences and Department of Chemistry, Ewha Womans University, Seoul, Republic of Korea.
authors.forename: Je Won
authors.initials: JW
authors.lastname: Park
authors.forename: Won Seok
authors.initials: WS
authors.lastname: Jung
authors.forename: Sung Ryeol
authors.initials: SR
authors.lastname: Park
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Park
authors.forename: Yeo Joon
authors.initials: YJ
authors.lastname: Yoon
mesh_terms: D039903:Actinobacteria / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000032:analysis; D001426:Bacterial Proteins; D002853:Chromatography, Liquid; D003065:Coenzyme A / Q000032:analysis*; D004952:Esters / Q000032:analysis*; D048630:Polyketide Synthases / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D004952:Esters; D048630:Polyketide Synthases; D003065:Coenzyme A