糖丝菌属次级代谢潜能分析及代表菌株基因编辑体系建立

Secondary metabolism potential of Saccharothrix and establishment of gene editing systems in representative strains

摘要:

【背景】稀有放线菌是发掘天然产物的“新矿藏”。糖丝菌(Saccharothrix)作为典型的稀有放线菌，其天然产物生产潜能尚需进行系统分析和发掘。此外，针对糖丝菌的基因编辑体系也鲜有报道。【目的】揭示糖丝菌属稀有放线菌合成不同结构类型天然产物的潜能，并建立代表菌株的基因编辑体系，推动新结构天然产物发现及相关生物合成研究。【方法】通过多位点序列分析(multi-locus sequence analysis,MLSA)方法评价已公布的34个糖丝菌基因组之间的相似度，利用antiSMASH分析基因簇及其合成产物的结构信息，并使用BiG-SCAPE对基因簇进行聚类分析。选择代表菌株澳大利亚糖丝菌(Saccharothrix australiensis) DSM 43800和紫丁香糖丝菌(Saccharothrix syringae) NRRL B-16468，以整合型载体和基因敲除载体为工具，建立接合转移及基因编辑体系。【结果】对34个糖丝菌基因组的分析显示，共发现了1 348个天然产物生物合成基因簇，平均每个基因组含有约40个基因簇。其中，合成聚酮、非核糖体肽、聚酮-非核糖体肽杂合产物，以及核糖体合成和翻译后修饰肽类天然产物的基因簇丰度较高。这1 348个基因簇聚类成852个基因簇家族(gene cluster family,GCF)，进一步聚集成130个基因簇集团(gene cluster clan,GCC)。本研究建立并优化了适用于澳大利亚糖丝菌和紫丁香糖丝菌的接合转移操作体系，并建立了2个代表菌株的基因编辑体系，获得了相应的突变菌株。【结论】糖丝菌作为稀有放线菌，其基因组内富含天然产物生物合成基因簇，展现出合成众多结构类型天然产物的强大潜能，尤其是聚酮与聚肽类天然产物。我们成功实现了对糖丝菌基因组的精准编辑，为深入研究基因簇及其合成的天然产物奠定了坚实的基础。

关键词:糖丝菌;次级代谢;天然产物;生物合成基因簇;基因编辑

Abstract:

[Background] Rare actinomycetes represent a “new treasure trove” for discovering natural products. Studies remain to be carried out to explore the natural product-producing potential of Saccharothrix as typical rare actinomycetes. Furthermore, few studies reported gene editing systems specifically tailored for Saccharothrix. [Objective] This study aims to elucidate the potential of Saccharothrix in synthesizing natural products with diverse structures. Additionally, we seek to establish gene editing systems for representative strains, thereby fostering the discovery of novel natural compounds and advancing the research on related biosynthetic pathways. [Methods] Multi-locus sequence analysis (MLSA) was employed to assess the similarity among 34 publicly available Saccharothrix genomes. The tool antiSMASH was utilized to analyze the gene clusters and the structural information of associated products. Additionally, BiG-SCAPE was applied for clustering analysis of these gene clusters. The representative strains, Saccharothrix australiensis DSM 43800 and S. syringae NRRL B-16468, were selected, for which the conjugation transfer and gene editing systems were established via integrative vectors and gene knockout vectors. [Results] The analysis of the 34 Saccharothrix genomes revealed a total of 1 348 natural product biosynthetic gene clusters, with an average of approximately 40 clusters per genome. The gene clusters were abundant in the biosynthesis of polyketides, non-ribosomal peptides, hybrid products of polyketides and non-ribosomal peptides, as well as ribosomally synthesized and post-translational modified peptides. The 1 348 gene clusters were grouped into 852 gene cluster families (GCFs), which were further grouped into 130 gene cluster clans (GCCs). This study established and optimized a conjugation transfer system applicable to S. australiensis DSM 43800 and S. syringae NRRL B-16468. Additionally, gene editing systems were successfully established for the two representative strains, with the establishment of corresponding mutant strains. [Conclusion] As rare actinomycetes, Saccharothrix exhibit a rich array of natural product biosynthetic gene clusters, highlighting robust potential for synthesizing diverse natural compounds, particularly polyketides and polypeptides. Moreover, this study achieves precise editing of the Saccharothrix genome, laying a solid foundation for probing into the gene clusters and associated products.

Key words:Saccharothrix sp.;secondary metabolism;natural products;biosynthetic gene clusters;gene editing