拟无枝酸菌属次级代谢潜能分析及代表菌株基因编辑体系建立

Secondary metabolic potential of Amycolatopsis and development of gene editing systems for representative strains

摘要:

【背景】 拟无枝酸菌属作为一类重要的稀有放线菌，是抗菌、抗癌类药物的重要来源，其合成天然产物的潜能尚待系统分析及发掘。【目的】 揭示拟无枝酸菌属不同结构类型次级代谢产物合成潜能，并建立两个代表性菌株的遗传操作体系，为结构新颖或生物活性优良的次级代谢产物发掘及研究提供条件。【方法】 使用多位点序列分析(multi-locus sequence analysis, MLSA)方法评价已公布的拟无枝酸菌基因组之间的相似度。同时，使用antiSMASH分析所有基因组的基因簇，预测其合成产物结构信息，并利用BiG-SCAPE对基因簇进行聚类分析。利用pSET152整合载体优化两个代表性拟无枝酸菌的接合转移条件，以建立遗传操作体系。采用CRISPR-cBEST碱基编辑和传统的同源重组确定靶基因失活方案。【结果】 146个拟无枝酸菌基因组的生物信息学分析显示，平均每个基因组中含有33个基因簇。其中聚肽、聚酮及萜类合成基因簇的丰度较高，并且大多数基因簇与已知化合物基因簇不同。成功确定了拟无枝酸菌属两个代表菌株的最适接合转移条件。尽管CRISPR基因编辑质粒能够转入拟无枝酸菌，但6个载体均未成功编辑目标基因，而利用传统同源重组方法则成功敲除目标基因，建立了目的基因敲除系统。【结论】 次级代谢潜能分析显示拟无枝酸菌属中蕴含丰富的新颖天然产物资源。此外，成功建立的基因编辑体系实现了外源基因整合及内源基因敲除，为该属新颖次级代谢产物的挖掘及生物合成研究提供了坚实的研究基础。

Abstract:

[Background] As an important group of rare actinomycetes, Amycolatopsis serves as a significant source of antimicrobial and anticancer drugs. The rich biosynthetic gene clusters (BGCs) of secondary metabolites in the genomes of Amycolatopsis await systematic analysis and mining. [Objective] To unveil the biosynthetic potential of secondary metabolites with different structural types in Amycolatopsis and establish genetic manipulation systems for two representative strains, laying a foundation for mining structurally novel or bioactive secondary metabolites. [Methods] The multi-locus sequence analysis (MLSA) was performed to evaluate the similarity among publicly available Amycolatopsis genomes. Simultaneously, antiSMASH was employed to analyze the gene clusters in all genomes and predict the structural information of the products. BiG-SCAPE was used for cluster analysis. The integration plasmid pSET152 was employed to optimize the conjugation transfer conditions for two representative Amycolatopsis strains, and then genetic manipulation systems were established. CRISPR-cBEST and homologous recombination were employed to determine the strategies for target gene inactivation. [Results] Bioinformatics analysis of 146 Amycolatopsis genomes revealed an average of 33 BGCs per genome. The BGCs associated with peptides, polyketides, and terpenes exhibited high abundance, with the majority differing from known BGCs. The optimal conjugation transfer conditions for two representative Amycolatopsis strains were successfully determined. Although CRISPR gene editing plasmids could be transferred into Amycolatopsis, none of the six vectors successfully edited the target gene. Conversely, target genes were successfully knocked out by homologous recombination, on the basis of which the gene editing systems were established. [Conclusion] The secondary metabolic potential unveils a rich repository of novel natural products within Amycolatopsis. Moreover, the successfully established genetic editing system enables the integration of exogenous genes and the inactivation of endogenous genes, laying a solid foundation for the mining and biosynthesis of novel secondary metabolites in this genus.