Genome mining of the mithramycin producer Streptomyces argillaceus ATCC 12956 revealed 31 gene clusters for the biosynthesis of secondary metabolites, and allowed to predict the encoded products for 11 of these clusters. Cluster 18 (renamed cluster arp) corresponded to a type I polyketide gene cluster related to the previously described coelimycin P1 and streptazone gene clusters. The arp cluster consists of fourteen genes, including genes coding for putative regulatory proteins (a SARP-like transcriptional activator and a TetR-like transcriptional repressor), genes coding for structural proteins (three PKSs, one aminotransferase, two dehydrogenases, two cyclases, one imine reductase, a type II thioesterase, and a flavin reductase), and one gene coding for a hypothetical protein. Identification of encoded compounds by this cluster was achieved by combining several strategies: (i) inactivation of the type I PKS gene arpPIII; (ii) inactivation of the putative TetR-transcriptional repressor arpRII; (iii) cultivation of strains in different production media; and (iv) using engineered strains with higher intracellular concentration of malonyl-CoA. This has allowed identifying six new alkaloid compounds named argimycins P, which were purified and structurally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Some argimycins P showed a piperidine ring with a polyene side chain (argimycin PIX); others contain also a fused five-membered ring (argimycins PIV-PVI). Argimycins PI-PII showed a pyridine ring instead, and an additional N-acetylcysteinyl moiety. These compounds seem to play a negative role in growth and colony differentiation in S. argillaceus, and some of them show weak antibiotic activity. A pathway for the biosynthesis of argimycins P is proposed, based on the analysis of proposed enzyme functions and on the structure of compounds encoded by the arp cluster.

CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.

Argimycins P are a recently identified family of polyketide alkaloids encoded by the cryptic gene cluster arp of Streptomyces argillaceus. These compounds contain either a piperideine ring, or a piperidine ring which may be fused to a five membered ring, and a polyene side chain, which is bound in some cases to an N-acetylcysteine moiety. The arp cluster consists of 11 genes coding for structural proteins, two for regulatory proteins and one for a hypothetical protein. Herein, we have characterized the post-piperideine ring biosynthesis steps of argimycins P through the generation of mutants in arp genes, the identification and characterization of compounds accumulated by those mutants, and cross-feeding experiments between mutants. Based in these results, a biosynthesis pathway is proposed assigning roles to every arp gene product. The regulation of the arp cluster is also addressed by inactivating/overexpressing the positive SARP-like arpRI and the negative TetR-like arpRII transcriptional regulators and determining the effect on argimycins P production, and through gene expression analyses (reverse transcription PCR and quantitative real-time PCR) of arp genes in regulatory mutants in comparison to the wild type strain. These findings will contribute to deepen the knowledge on the biosynthesis of piperidine-containing polyketides and provide tools that can be used to generate new analogs by genetic engineering and/or biocatalysis.