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Strain Improvement Of Streptomyces Lydicus And Investigation Of The Streptolydigin Biosynthesis

Posted on:2007-09-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:X B LiFull Text:PDF
GTID:1101360212970802Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
The comparative LC/ESI–MS analysis was successfully used for the characterization of reasons resulting in the yield increase in the selected mutant after strain improvement of Streptomyces lydicus AS 4.2501, and the biosynthetic pathway of streptolydigin was investigated by the LC/MS/MS analysis of culture extracts of this parent strain and its mutants. The results of this study were as follows:Isolated agar media plus propionate, glucose and valine with MIC were used to screen the precursor-tolerant/resistant mutants, and the positive frequency of 83.5% was obtained. Especially, a propionate-resistant mutant P28 showed an increase of 267% in streptolydigin titer over the starting strain S. lydicus AS 4.2501. In order to characterize the metabolic variations in intermediates, metabolites and biosynthetic pathways, culture extracts of the mutant P28 and its parent strain were parallel analyzed by LC/ESI–MS following the strain improvement of S. lydicus AS 4.2501. The results showed that two branch pathways blocked in the mutant and the accumulation of an intermediate decreased dramatically in the mutant cultures resulted in the streptolydigin overproduction in the mutant P28. Moreover, the compound at m/z 363.1 [M– H]ˉwas a key and significant intermediate being both the final product of polyketide biosynthesis and starting compound of tetramic acid moiety biosynthesis.In order to investigate the biosynthetic pathway of streptolydigin, the culture extracts using four strains S. lydicus AS 4.2501, P28, L8 and DSM 40461 were used for the LC/MS/MS analysis. Three possible pathways for the streptolol moiety and four for the tetramic acid moiety were proposed on the basis of the previous studies. Whether the hypothetical compounds existed in the culture extracts or not were decided by the combination of the extracted ion chromatogram (EIC) and ESI–MS scans on the corresponding molecular masses, thus, the unexpected pathways were obviously excluded and the exact pathway could be rapidly distinguished. The subsequent MS/MS or MS3 experiments provided direct evidences for the structural identification of the proposed pathway intermediates, via their substructural breakpoints as well as fragment ions along with structural origins...
Keywords/Search Tags:Streptolydigin, Streptomyces lydicus, Strain improvement, Biosynthetic pathways, LC/MS analysis, Metabolites, Structural identification
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