Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry.
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.
Park
Je Won
JW
Division of Nano Sciences and Department of Chemistry, Ewha Womans University, Seoul, Republic of Korea.
Jung
Won Seok
WS
Park
Sung Ryeol
SR
Park
Byoung Chul
BC
Yoon
Yeo Joon
YJ
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
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D004952:Esters; D048630:Polyketide Synthases; D003065:Coenzyme A