环圈链霉菌(Streptomycesanulatus) 89-2-2全基因组测序及序列分析

Whole-genome sequencing and sequence analysis of Streptomyces anulatus 89-2-2

摘要:

【背景】环圈链霉菌(Streptomyces anulatus) 89-2-2是一株中度嗜盐放线菌，其次级代谢产物具有抑制蘑菇酪氨酸酶活性、抑制小鼠黑色素瘤B16细胞内酪氨酸酶活性及黑色素合成的作用。目前S. anulatus 89-2-2基因组序列分析尚未见报道，这限制了该菌株中酪氨酸酶抑制剂、黑色素合成抑制剂及其他次级代谢产物合成及调控的研究。【目的】解析S. anulatus 89-2-2基因组序列信息，挖掘其次级代谢产物基因资源，为深入研究该菌株的酪氨酸酶抑制剂产生机理及生物合成调控机制奠定基础。【方法】利用Nanopore测序平台对S. anulatus 89-2-2进行全基因组测序；采用相关软件对菌株89-2-2的基因组序列进行拼接、基因预测、功能注释、进化分析和共线性分析，并预测次级代谢产物合成基因簇，比较不同链霉菌的基因组间差异，研究不同种链霉菌的次级代谢潜力及物种间的进化关系。【结果】菌株89-2-2基因组为一条总长度为8 117 999 bp的线性染色体，G+C含量为71.52%，序列已提交至NCBI的GenBank数据库，登录号为CP137002。生物信息学分析发现基因组含有7 088个编码基因，分别在COG、GO、KEGG、NR数据库提取到5 300、4 176、2 513、7 013个基因的注释信息。同时，通过antiSMASH软件预测到34个次级代谢产物合成基因簇，涉及萜烯类、非核糖体肽类、聚酮类、翻译后修饰肽类等多种天然产物的合成，其中11个基因簇与已知化合物编码基因簇同源性较低，说明该菌株具有产生多种新型次级代谢产物的潜力。通过对菌株89-2-2与S. microflavus DSM40593、Streptomyces sp. AM2-1-1基因组比较分析发现，菌株89-2-2与菌株AM2-1-1基因组线性关系较好，具有较近的亲缘关系；而与菌株DSM40593存在较大区域的倒位现象，亲缘关系较远。菌株89-2-2特有的基因家族和基因数目分别为51个和1 235个。这些结果表明，链霉菌属具有较强的适应多样生态环境的能力，在长期适应过程中，种间的基因组出现了局部的波动，菌株89-2-2特有基因与不同生境的适应关系还需要进一步研究。【结论】分析了菌株89-2-2的全基因组信息，为探究该菌株抑制酪氨酸酶活性与基因组间的联系、分析其次级代谢产物合成基因簇的结构、评估链霉菌次生代谢潜力奠定了基础，为新颖化合物的发现和新药物的开发提供了依据。

关键词:中度嗜盐链霉菌;酪氨酸酶抑制剂;全基因组测序;生物合成基因簇

Abstract:

[Background] Streptomyces anulatus 89-2-2, a moderately halophilic actinomycete strain, can produce secondary metabolites capable of inhibiting the tyrosinase activity in mushrooms and the melanin synthesis and tyrosinase activity in mouse melanoma B16 cells. Few studies report the genome sequence of S.anulatus 89-2-2, which limits the studies on the biosynthesis and regulation of tyrosinase inhibitors, melanin synthesis inhibitors and other secondary metabolites in the strain. [Objective] This study sequenced the genome of S.anulatus 89-2-2 and mined the genetic resources of secondary metabolites, aiming to lay a foundation for deciphering the mechanisms of tyrosinase inhibitor production and biosynthesis regulation in this strain. [Methods] Nanopore sequencing platform was used to uncover the genome sequence of S. anulatus 89-2-2. Bioinformatics tools were used for sequence assembly, gene prediction, functional annotation, phylogenetic analysis, synteny analysis, and prediction of the gene clusters for biosynthesis of secondary metabolites. The genomes of different Streptomyces spp. were compared, and the secondary metabolic potential and the evolutionary relationship of different Streptomyces spp. were studied. [Results] The genome of strain 89-2-2 was a single linear chromosome with a length of 8 117 999 bp and the G+C content of 71.52%. The sequence has been submitted to the GenBank of the NCBI, with the accession number CP137002. The genome of the strain contained 7 088 coding sequences. The annotation against COG, GO, KEGG, and NR predicted 5 300, 4 176, 2 513, and 7 013 genes, respectively. The antiSMASH predicted 34 gene clusters for the biosynthesis of secondary metabolites in the genome of 89-2-2, and these gene clusters were involved in the biosynthesis of a variety of natural products, such as terpenoids, non-ribosomal peptides, polyketides, and ribosomally synthesized and post-translationally modified peptides. Eleven clusters showed low similarities to the gene clusters for the biosynthesis of known compounds, which suggested that strain 89-2-2 had the potential to produce a variety of novel secondary metabolites. The genome of S.anulatus 89-2-2 presented high synteny with that of Streptomyces sp. AM2-1-1, which indicated their high homology. It showed large inversions compared with that of S.microflavus DSM40593, which suggested their low homology. Strain 89-2-2 had 51 specific gene families and 1 235 specific genes. The results indicated that Streptomyces spp. had strong ability to adapt to diverse environments, and partial fluctuations in the genomes occurred between species during the long-term adaptation. Further studies remain to be carried out to reveal the relationship between specific genes and adaptability of strain 89-2-2 to different environments. [Conclusion] This study analyzed the whole-genome sequence of S.anulatus 89-2-2, laying a foundation for further studies about the strain in terms of the association between the tyrosinase-inhibiting activity and the genome, the structures of gene clusters for the biosynthesis of secondary metabolites, and the secondary metabolic potential of Streptomyces spp. This study provides basic data for the discovery of novel compounds and new drugs.

Key words:moderately halophilic Streptomyces spp.;tyrosinase inhibitor;whole-genome sequencing;gene cluster for biosynthesis