Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

Actinomycetes are remarkable producers of compounds essential for human and veterinary medicine as well as for agriculture. The genomes of those microorganisms possess several sets of genes (biosynthetic gene cluster (BGC)) encoding pathways for the production of the valuable secondary metabolites. A significant proportion of the identified BGCs in actinomycetes encode pathways for the biosynthesis of polyketide compounds, nonribosomal peptides, or hybrid products resulting from the combination of both polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The potency of these molecules, in terms of bioactivity, was recognized in the 1940s, and started the “Golden Age” of antimicrobial drug discovery. Since then, several valuable polyketide drugs, such as erythromycin A, tylosin, monensin A, rifamycin, tetracyclines, amphotericin B, and many others were isolated from actinomycetes. This review covers the most relevant actinomycetes-derived polyketide drugs with antimicrobial activity, including anti-fungal agents. We provide an overview of the source of the compounds, structure of the molecules, the biosynthetic principle, bioactivity and mechanisms of action, and the current stage of development. This review emphasizes the importance of actinomycetes-derived antimicrobial polyketides and should serve as a “lexicon”, not only to scientists from the Natural Products field, but also to clinicians and others interested in this topic.

Kirromycin is the main product of the soil-dwelling Streptomyces collinus Tü 365. The elucidation of the biosynthetic pathway revealed that the antibiotic is synthesised via a unique combination of trans-/cis-AT type I polyketide synthases and non-ribosomal peptide synthetases (PKS I/NRPS). This was the first example of an assembly line integrating the three biosynthetic principles in one pathway. However, information about other enzymes involved in kirromycin biosynthesis remained scarce. In this study, genes encoding tailoring enzymes KirM, KirHVI, KirOI, and KirOII, and the putative crotonyl-CoA reductase/carboxylase KirN were deleted, complemented, and the emerged products analysed by HPLC-HRMS and MS/MS. Derivatives were identified in mutants ΔkirM, ΔkirHVI, ΔkirOI, and ΔkirOII. The products of ΔkirOI, ΔkirOII, and kirHVI were subjected to 2D-NMR for structure elucidation. Our results enabled functional assignment of those enzymes, demonstrating their involvement in kirromycin tailoring. In the ΔkirN mutant, the production of kirromycin was significantly decreased. The obtained data enabled us to clarify the putative roles of the studied enzymes, ultimately allowing us to fill many of the missing gaps in the biosynthesis of the complex antibiotic. Furthermore, this collection of mutants can serve as a toolbox for generation of new kirromycins.

Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism. This new trend has been accompanied with development of new computational resources (genome and compound mining tools), generation of various high-quality omics data, establishment of molecular tools, and other strain engineering strategies. They all come together to enable systems metabolic engineering of streptomycetes, allowing more systematic and efficient strain development. In this review, the authors present recent progresses within systems metabolic engineering of streptomycetes for uncovering their hidden potential to produce novel compounds and for the improved production of secondary metabolites.

Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes.

Filamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, singlenucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.3 MainMore than 70% of current antibiotics are derived from natural products of actinomycetes. Genome mining indicates that these organisms still possess a huge unexploited potential of producing our future antimicrobial drugs 1 . However, for exploiting this potential, modern bio-technologies, such as metabolic engineering and synthetic biology, are heavily relying on efficient genetic manipulation or gene editing approaches 1 . Unfortunately, it is relatively difficult to do genome manipulation of actinomycetes, mainly due to their mycelial growth, intrinsic genetic instability and very high GC-content (>70%) of their genomes. There are established traditional mutagenesis methods, but they are relatively inefficient and very time-and laborconsuming 2 .Recently, more efficient CRISPR-Cas9 systems were developed for scar-less gene knockout, knockin and reversible gene knockdown in actinomycetes 3 . Although these systems provide excellent flexibility and high efficiency, severe challenges still remain.In many actinomycetes, the (over)expression of Cas9 has severe toxic effects and leads to a high number of unwanted off-target effects 3 . Furthermore, the linear chromosomes show a relatively high intrinsic instability and can tolerate large-scale chromosomal deletions and rearrangements 4 . DNA double-strand breaks (DSB) in the arm region are considered major triggers of this instability 5 and often co-occur with the mutagenesis procedures.Here, we present an alternative highly efficient approach to generate mutations in filamentous actinomycetes without the requirement of DSBs. The targeted conversion of cytidine (C) to thymidine (T) can lead to the introduction of stop codons 6-9 and lossof-function mutations into the coding genes of different organisms. In particular, we also can introduce rare TTA codons to artificially put genes under BldA control 10 . Such