Study On Secondary Metabolites Produced By Two Strains Of Soil Streptomyces And Functional Characterization Of Pyridomycin Biosynthtic Gene Pyr2

Actinomycetes, especially streptomyces, have distinguishedadvantages in the production of antibiotics. Many antibiotics that havebeen used widely today were produced by the streptomyces isolated fromsoil. Furthermore, the materials are not problematic any more thanks tolarge-scale fermentation. In the previous research, two strains of thestreptomyces YIM48915and YIM48924were isolated from forest soil inthe southwest china. The strain YIM48915can inhibit the growth ofStaphylococcus aureus, Streptomyces lividans TK24and Rhodotorulaglutinis. Firstly, we optimized the fermentation condition based on theUV-vis spectra of the secondary metabolites. Secondly,20L fermentationwas performed. Finally，Four compounds from YIM48915and one fromYIM48924were isolated and purified by the liquid-liquid extraction,column chromatography, and reverse HPLC preparation. The structures ofthe four compounds from YIM48915were elucidated by1D-NMR,2D-NMR and MS spectra. Compound15-1is identified as6,7-dichloro-1-methyltryptophan, a new compound. The compound15-2isRabelomycin, a member of Angucycline family. The compound15-3isdetermined to be a member of the β-Carboline family. The compound15-5is a derivative of the compound15-1,6,7-dichloro-1-methyl-1H-indole-3-carboxyl acid. Thus, the YIM48915strain can produce the secondary metabolites with structural diversities.My thesis also includes the preliminary study on the function of the gene pyr2in the biosynthesis of pyridomycin. The LC-MS analysisindicated that a precursor to pyridomycin was produced by the inactivationmutant of pyr2. The compound HTT12-1was purified and structurallyelucidated by using spectroscopic techniques. In addition, other twocompounds HTT12-2and HTT12-3were purified and the structure ofHTT12-2was elucidated. HTT12-2was a product of HTT12-1that lost thelast moiety of2-hydroxy-3-methylpentanoic acid. After identification ofHTT12-1, we carried out a biochemical assay with the purified Pyr2andNADPH as a cofactor. However, we did not detect any expected peaksfrom LC-MS analysis. We proposed two possibilities: the one is thatHTT12-1is not real substrate of Pyr2; the other one is that the assaycondition is not suitable for Pyr2-catalyzed reaction. Anyway, theidentification of HTT12-1and HTT12-2provided the opportunity toinvestigate the mechanism of the formation of the last2-hydroxy-3-methylpent-2-enoic acid moiety in pyridomycin biosynthesis.