Study And Application On Metabolomic Profiling Of Streptomyces Albus Based On LC-MS

Streptomyces has exceedingly strong secondary metabolites and quite diverse metabolic forms.It provides rich natural products which is widely used in human medicine,animal health,and plant crop protection.However,our comprehensions of the metabolic pathway of Streptomyces and the function of the biosynthetic gene cluster is relatively limited.Based on the analysis of microbial metabolomics,we can study the cellular functions of Streptomyces further more.(1)Currently,the identification of metabolites has become one of the most important limiting factors in the development of metabolomics.The development of metabolomics based on LC-MS can be effectively promoted by establishing a massspectral tag library.We detected the metabolites of S.albus by HPLC-Q-TOF,and identified metabolites structures by auto ms/ms to establish a basic MS~2 spectral tag library of S.albus metabolites.First,we obtained a total of 297(almost)non-redundant metabolites with MS~2 spectra and identified 52 of them.The database lays the foundation for our subsequent analysis of metabolic differences and functional gene cluster analysis of biosynthesis.(2)To reveal the biosynthetic pathway of SF2768 expressed by S.albus and search for possible biosynthetic precursors and explain the function of biosynthetic gene cluster,we compared the metabolism differences between wild-type S.albus and SF2768 heterologous expression mutants(S.albus::pZMY13C).To search of the possible precursors of SF2768,we used the metabolomic multi-group comparison method(Meta XCMS job)to analyze metabolomic differences in four gene disruption strains of?sfaA,?sfaB,?sfaC,?sfaD and the heterologous expression strain S.albus::pZMY13C(The previous studies showed that?sfaA,?sfaB,?sfaC,?sfaD and?sfaE completely did not produce antibiotic SF2768).Through the experiments,we have found that glutamic acid,histidine,tyrosine,phenylglycine,aminobutyric acid and niacin were the key metabolites in the biosynthetic pathway of SF2768.To further confirmed our results,we added these 6 compounds during the fermentation of S.albus::pZMY13C and examined the production changes of SF2768.Simultaneously adding equal amounts of 6possible precursors,phenylglycine greatly up-regulated the production of SF2768,most likely the precursor of biosynthesis of SF2768.(3)We have analyzed the metabolic differences between the wild type strains and the corresponding heterologous expression strain through the established methods.The strains included S.albus,S.albus::pZAY5B1,S.albus::pZAY10A3,S.albus::pZAY14B8,S.albus::pZAY23F11,and they produced antibiotics such as enteromycin and holomycin.We detected the target metabolites and analyzed the differences in metabolism between the wild strain and the heterologous expression strains by LC-MS.Finally,we found the key metabolites that affected the heterologous expression.The mass spectral database of key metabolites was established through the metabolomic analysis of S.albus with HPLC-HRMS.The differences of metabolites of heterologous expression strains with S.albus as host were analyzed.It is of great significance in the functional analysis of biosynthetic gene cluster and the discovery of new antibiotics and other studies.