Isolation And Identification Of Streptomyces Sp.XZHG99~T And Its Secondary Metabolites

Nowadays,the problem of bacterial resistance is more and more serious,which makes the existing antibiotic treatment being faced with enormous challenges.It is found that bioprospecting on microbial medicinal resources from special habitats including desert is an effective approach to develop new antibiotics.In this study,the strain Streptomyces sp.XZHG99 producing abundant secondary metabolites were found through bioprospecting and biological activity screening of the actinomycetes from color desert in Dengpa,Tibet.Then the strain XZHG99 was identified,and its secondary metabolites were purified and analyzed.Lastly,the ribosome engineering was performed for improving production of antibiotics.The main results are listed as follows:16S rRNA gene analysis revealed the strains isolated from color desert in Dengpa were affiliated with seven actinobacterial genera.Streptomyces was the dominant genus.Screening of biosynthetic gene clusters showed that 68 strains had at least one of four biosynthetic gene clusters and 6 strains had all the tested biosynthetic gene clusters.Furthermore,secondary metabolites produced by these strains showed antimicrobial activity against certain pathogens and antioxidant capacity towards hydroxyl and oxygen free radical scavenging.Therein,eight strains displayed broad-spectrum antimicrobial activity.Thirteen strains showed the positive total antioxidant activity.Ten strains had good hydroxyl free radical scavenging and three strains showed positive oxygen free radical scavenging.LC-MS analysis of secondary metabolites from some strains showed that the secondary metabolite profile of strain XZHG99was the most abundant.The strain XZHG99 was identified by 16S rRNA sequence analysis,morphological and cultural characterization,physiological and biochemical characterization,and chemotaxonomic features.The type strain XZHG99 was deposited and its type strain number is XZHG99^T.The results are listed as follows:The 16S rRNA gene sequence analysis indicated that strain XZHG99^T showed high similarity to S.albiflavescens m20^T?98.42%?and S.krungchingensis KC-035^T?98.14%?,and the DNA-DNA relatedness between strain XZHG99^T and its reference strains were well below the threshold of 70%for delineation of genomic species.These data suggested that strain XZHG99^T was considered to represent a novel species of the genus Streptomyces.Furthermore,strain XZHG99^T was determined to represent a novel species of the genus Streptomyces accrodingt to the characteristics capable of distinguishing from its reference strains including morphological,physiological,biochemical and chemotaxonomic characteristics,for which the name Streptomyces dengpaensis sp.nov.is proposed.The optimal medium for S.dengpaensis XZHG99^T producing secondary metabolites was Starch-Casein medium containing 3%sea salt.The large-scale fermentation using above medium.Then the ethyl acetate extract of the secondary metabolite was collected and applied to separation,purification and structure analysis of compound.Nine compounds were obtained,numbered 1⁹,which were composed of two known actinomycins and seven Angucyclines including three new Angucyclines?grincamycins L?1??,grincamycins M?2?,grincamycins N?3?).Compounds 1-9 showed low cytotoxicity against tumor cell lines including human lung cancer cells?A549,H157?,human breast cancer cells?MCF7,MDAMB231?and human hepatoma cells?HepG2?with IC₅₀ values being ranging from 0.09 nM to 17.30?M.Compounds 4,8 and 9 have good antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus with IC₅₀ values being ranging from 0.12?M to 23.1?M.The wild S.dengpaensis XZHG99^T was used for ribosomal engineering breeding.The resulting rpsL gene mutants HTT6 and HTT8 showed higher production of Angucyclines then the others.The colonies of HTT6 and HTT8 become smaller,decreased sporulation ability,improved yields of Angucyclines,and were less susceptible to the medium.Comparing with the wild strain,the yield of rabelomycin produced by HTT6 was improved to be 2.5 to 7.7 times,and the production of saquayamycin B1 was improved to be 2 to 32 times.The yield of rabelomycin produced by HTT8 was 1.8 to 13 times that of the wild strain,and the yield of saquayamycin B1 was improved to be 2.5 to 45 times.