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.
