内生真菌HND5菌株抗香蕉枯萎病菌相关非核糖体多肽合成酶基因簇的鉴定

Identification of the non-ribosomal peptide synthetase(NRPS) gene clusters involved in resistance to Fusarium oxysporum f. sp. cubense in the endophytic fungal isolate HND5

为明确帚枝霉属内生真菌Sarocladium brachiariae HND5菌株具体的抑菌活性物质,在全基因组测序数据的基础上,利用生物信息学对抑菌活性物质合成基因簇进行定位,通过聚类分析和系统发育树分析对基因簇合成产物进行鉴定,并通过基因缺失突变构建及代谢组分析确定具体的抑菌活性物质。结果显示,HND5菌株共含有7个非核糖体多肽合成酶(non-ribosomal peptide synthetase,NRPS)基因簇,聚类分析结果表明基因簇29合成产物为噬铁素,基因簇30合成产物为类环孢霉素类抗菌多肽;系统发育树结合生物信息学结果显示基因簇30合成产物与已知环孢霉素结构差异大,为一个新型非核糖体多肽;将基因簇30 NRPS基因缺失突变后,HND5菌株丧失抑菌活性;同野生型菌株相比,基因簇30 NRPS基因缺失突变体缺失一个分子量为887.54 Da的多肽类物质。表明HND5菌株的抑菌活性物质为一个分子量为887.54 Da的新型类环孢霉素非核糖体多肽,基因簇30负责该多肽的生物合成。

In order to identify the antifungal substances of the endophytic fungus Sarocladium brachiariae HND5 strain, bioinformatic methods were used to locate putative genes responsible for the biosynthesis of antifungal substances based on the whole genome sequence of this strain, and cluster analysis combined with phylogenic analysis was used to identify the products of gene clusters and the exact antifungal substance was located by constructing deletion mutants and analyzing metabolome. The results showed that HND5 strain had seven non-ribosomal peptide synthetase(NRPS) gene clusters;the product of cluster 29 was siderophore and the product of cluster 30 was cyclosporin-like non-ribosomal antifungal peptide based on cluster analysis. Phylogenetic analysis combined with bioinformatic analysis indicated that the product of cluster 30 was a new non-ribosomal peptide, the structure of which was very different from cyclosporin. HND5 strain lost its antifungal activity when cluster 30 NRPS gene was mutated by deletion. Compared with HND5 wild type strain, the deletion mutant of cluster 30 NRPS gene lost an 887.54 Da peptide. This study suggested that HND5 strain synthesized an 887.54 Da cyclosporin-like non-ribosomal peptide by gene cluster 30 as the antifungal substance.