Study On Secondary Metabolites Of A Halophilic

One of the hottest natural medicine research is to looking for novel bioactive compounds from the secondary metabolites which produced by microorganisms living in extreme environments. Halophilic bacteria as the significant microbial resource, the special living environment gives its unique physiological structure、metabolic mechanism and rich functional genes. In recent years, lots of new species halophilic bacteria are discovered. Whether the filamentous spore halophilic actinomycetes which produce natural products and their derivatives with a new structure can become of the new bacteria source? That is worth to exploring.In this research, 60 strains of halophilic bacteria used in our study were isolated from Aiding lake and Qijiaojing salt lake, Including 58 filamentous spore halophilic actinomycetes and two filamentous spore halophilic archaea. In 450 mL little fermentation, different strains used different culture medium and salt concentration to screening of fermentation conditions and experimental strains. The fermentation broth was extracted by EtOAc, the crude extract were analyzed by TLC for chemical diversity. According to the analysis results, we selected from the 5 strains of halophilic bacteria, and expand the amount of fermentation to 20 L. The fermentation conditions were consistent with the previous. The crude extract which produced by the 5 strains were initial separated and purified, and analyzed by TLC for chemical diversity. It was found that the TLC chemical diversity of secondary metabolites had some differences compared with the preliminary screening, and the diversity of the secondary metabolites was less than before. YIM 93972 and YIM 93974 are two special strains which have an important taxonomic and morphological differentiation of importance, they are the unique halophilic archaea which can produce substrate mycelium, aerial hyphae, long spore chains and spores(currently, all the archaea don’t have the capacity to produce any substrate mycelium, aerial hyphae, long spore chains and spores). Finally, we chose YIM 93974 which provided with rich and stable metabolite as the target strain to amplify fermentation.The total amount of fermentation of strain YIM 93974 was 210 L. After extracting with an equal volume of EtOAc, we obtained crude extract 11 g. Using silica gel column chromatography, gel chromatography and HPLC methods for the separation and purification of crude extract. 45 compounds were isolated from the strain YIM 93974 by varied chromatographic methods. There are six small amount of those compounds which did not get MS and NMR spectral data. Through data analysis and structure identification, we identified the structure of 28 compounds. These compounds are mainly divided into four categories: cyclic peptides, small molecule phenols, nucleic acid and indoles. There are 10 of cyclic peptides, such as Cyclo-(L-tryptophyl-L-proline), Cyclo-(L-prolyl-D-valine) and Cyclo-(L-prolyl-L-valine). There are 5 of small molecule phenols, such as Parahydroxybenzaldehyde, 1-(4-Hydroxyphenyl) ethanone and 2-Hydroxybenzyl alcohol. There are 6 of nucleic acid, for instance 5’-O-Methyladenosine, 2,4-Dihydroxypyrimidine and 2,4-Dihydroxy-5-methylpyrimidine. In addition, there are 7 other classes of compounds, for example, 3-(hydroxyacetyl) indole; Indole-3-carboxaldehyde; 3-Indolylacetamide. The results showed that the largest number of metabolites were cyclic peptide compounds for strain YIM 93974, and the cyclic peptide compounds were the representative metabolites.The results showed that the 58 halophilic actinomycetes can produce rich secondary metabolites. They are an important source of natural products and their derivatives, and has important research value. However, the stability of their metabolites in fermentation needs further study. YIM 93974 as a unique strain of Streptomyces-like archaea, although the study found that a large number of compounds was produced, but these compounds are almost the small molecule compounds, and YIM 93974 have no phenomenon to produce the same rich secondary metabolites as Streptomyces. The fermentation conditions are not suitable or bacteria do not have this capability, we need in-depth study of metabolomics combined with genome to further validated in the future.