Differential analysis of secondary metabolites by LC-MS following strain improvement of Streptomyces lydicus AS 4.2501.
Metabolite variations in a high-yielding mutant and its parent strain were studied by comparative LC-MS analysis after strain improvement. Streptomyces lydicus AS 4.2501-P28, a propionate-resistant mutant isolated by the high-frequency screening method using the principle of eliminating precursor inhibition effects, showed an increase of 267% in streptolydigin titre over the starting strain. Culture extracts of this mutant and its parent strain were analysed in parallel by an LC-MS technique, including full scan and extracted-ion scan, ESI-MS (electrospray-ionization MS) detection, DAD (diode-array detection) and MS2 (tandem MS) measurement. The main metabolic variations were obviously found in intermediates, metabolites and biosynthetic pathways: two unknown metabolites with the molecular [M-H]- ions at m/z 423.3 and 687.2, corresponding to two branch pathways, were blocked in the mutant, and the accumulation of a significant intermediate at m/z 363.1 [M-H]- decreased dramatically in the mutant cultures, resulting in the overproduction of streptolydigin (an antibiotic that inhibits prokaryotic RNA polymerase) in the mutant. Ion fragmentations of the tandem-MS spectra provided experimental evidence for the structural characterization of the three compounds obtained. In comparison with the traditional methods, comparative LC-MS analysis was rapid, sensitive and suitable for characterizing intermediates, metabolites and pathways for elucidation of the metabolic alterations after the isolation of improved strains.
Li
Xiao-Bing
XB
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China.
Qiao
Bin
B
Yuan
Ying-Jin
YJ
D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D002853:Chromatography, Liquid; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000494:pharmacology; D013058:Mass Spectrometry; D008954:Models, Biological; D016296:Mutagenesis; D011422:Propionates / Q000494:pharmacology; D013302:Streptomyces / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014633:Valine / Q000494:pharmacology
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D017077:Culture Media, Conditioned; D011422:Propionates; C100307:streptolydigin; D014633:Valine; D005947:Glucose