| Actinomycetes have a huge reserve in nature,and they are excellent carriers of natural active compounds,especially Streptomyces,which have synthesized most of the known natural antibiotics.In this study,actinomycete TR19040 was isolated from four soil samples collected from Xinglong Mountain,Gansu Province.It has the characteristics of abundant secondary metabolites,easy separation and high research value.In addition,its microbial community had good antagonistic activity against Fusarium graminearum and Fusarium proliferatum.And the experiment found that under the optimal culture condition of actinomycete TR19040,when the fermentation amount was 40 ml,3.83 mg of secondary metabolites could be extracted,which fully meets the requirements of this study.The DNA sequence of TR19040 was successfully extracted in this study.Then,the phylogenetic tree was constructed by neighbor joining and the strain was identified as Streptomyces in actinomycetes.The results of sequencing the whole gene of actinomycete TR19040 showed that the GC content of the strain was 72.56%,and 7389 coding genes were found.The protein sequence was compared in COG database,and 214 genes were related to the biosynthesis of secondary metabolites in TR19040.Meanwhile,the types of biosynthetic gene clusters in actinomycete TR19040 were labeled and the corresponding natural products were predicted.The single gene functions with high homology with known genes in gene clusters region 9.1,region 67.1 and region 97.1 were analyzed.It is proved that TR19040 has the potential to synthesize many natural products,especially the iron carrier,phenols and polyketones.This may be the first time to study the whole gene sequence of actinomycete TR19040 in detail.Based on the prediction of the biosynthetic potential of strain TR19040,the 7.86 g secondary metabolites were isolated and purified.Finally,three polyketones,two polyketo-pyranonaphthoquinones,two terpenoids,one gliotoxin and one pentadecanoate were obtained.The structure type is consistent with the gene prediction,and they are all known structures.In the biological activity test,the compound gliotoxin(4)showed strong cytotoxic activity on A549,HL-60,SW480,MCF-7 and SMMC-7721.The IC50 values ranged from0.11 to 1.45 μM.The inhibition effect of pyranophthoquinone A(5)on SMMC-7721,MCF-7and SW480 was weak,and inhibitory effect on SMMC-7721 was better than that of pyranophthoquinone B(6).At the same time,compound 5 was the best in the evaluation of NO generation inhibition activity,and the inhibition rate was 24.79±0.28%.In addition 6 has the cytotoxic activity second to 4 in the experiment.It has half inhibition rate on MCF-7,HL-60 and SW480 human cancer cells in vitro,IC50 values ranged from 3.5-13.2 μM.At the same time,the inhibition rate of NO production to RAW264.7 cells was 11.22 ± 2.30%.Besides,aloesaponarin(2)also has a certain inhibition effect on NO production and cytotoxic effect,while other products have not recorded obvious inhibitory activity.This paper is the first time to test the anti-cancer activity of natural products 5 and 6 by five human cancer cells,and also the first time to evaluate their anti-inflammatory activity.Then,the potential of their proprietary medicine was discussed,which filled the gap of related research.Moreover,the chemical structure of modified 5 was explored,and the results are of great significance for reference.This study combines theory with practice.The technology of separating and protecting bacteria,molecular biology technology,natural product separation technology and limited organic synthesis technology are fully used.The first comprehensive study of actinomycete TR19040 was completed.Every stage of the experiment is valuable,and the results have room for discussion.This will contribute to the research of biosynthetic antibiotics in China. |
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