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The design and engineering of secondary metabolite gene clusters that are characterized by complicated genetic organization, require the development of collections of well-characterized genetic control elements that can be reused reliably. Although a few intrinsic terminators and RBSs are used routinely, their translation and termination efficiencies have not been systematically studied in Actinobacteria. Here, we analyzed the influence of the regions surrounding RBSs on gene expression in these bacteria. We demonstrated that inappropriate RBSs can reduce the expression efficiency of a gene to zero. We developed a genetic device – an in vivo RBS-selector – that allows selection of an optimal RBS for any gene of interest, enabling rational control of the protein expression level. In addition, a genetic tool that provides the opportunity for measurement of termination efficiency was developed. Using this tool, we found strong terminators that lead to a 17–100-fold reduction in downstream expression and are characterized by sufficient sequence diversity to reduce homologous recombination when used with other elements. For the first time, a C-terminal degradation tag was employed for the control of protein stability in Streptomyces . Finally, we describe a collection of regulatory elements that can be used to control metabolic pathways in Actinobacteria.

Background Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. Results In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans -based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp . chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. Conclusion The constructed S. lividans -based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Actinobacteria isolated from unstudied ecosystems are one of the most interesting and promising sources of novel biologically active compounds. Cave ecosystems are unusual and rarely studied. Here, we report the isolation and characterization of ten new actinobacteria strains isolated from an ancient underground lake and moonmilk speleothem from the biggest conglomeratic karstic cave in Siberia with a focus on the biological activity of the obtained strains and the metabolite dereplication of one active strain. Streptomyces genera isolates from moonmilk speleothem demonstrated antibacterial and antifungal activities. Some of the strains were able to inhibit the growth of pathogenic Candida albicans.

The phylum Actinomycetota and genus Streptomyces in particular are the major source for discovery of natural products with diverse chemical structures and a variety of biological activities. Genes encoding biosynthetic pathways for bacterial natural products are grouped together into biosynthetic gene clusters (BGCs). The size of a typical actinobacterial BGC may range from 10 kb to 200 kb, which makes their cloning for heterologous expression a challenging task. Various DNA cloning and assembly methods have been established for capturing BGCs. Among them, the transformation‐associated recombination (TAR) in Saccharomyces cerevisiae remains one of the most cost‐effective, accessible, customisable and precise approaches. However, the drawback of TAR cloning is a need for intensive screening of clones in order to identify one carrying the BGC. In this study, we report a further development of the TAR cloning approach by introducing the direct selection of colonies with BGC of interest based on the yeast killer phenomenon. For this, a new TAR cloning vector system was constructed and the strategy was validated by successful cloning of chelocardin (35 kb) BGC from Amycolatopsis sulphurea and daptomycin BGC (67 kb) from Streptomyces filamentosus. Both BGCs were functionally expressed in a heterologous host, resulting in the production of the corresponding antibiotics. The proposed approach could be widely applied for precise direct cloning of BGCs from the representatives of phylum Actinomycetota and easily adopted for other bacteria. Yeast killer toxin K1 was used as a counterselectable marker to improve the efficiency of TAR cloning procedure. The K1 toxin cassette‐based TAR cloning system was constructed and tested by capturing the chelocardin (35 kb) and daptomycin (67 kb) biosynthetic gene clusters.

Streptomyces viridosporus (formerly ghanaensis ) ATCC14672 is the best-studied producer of moenomycins, a family of phosphoglycolipid natural products directly inhibiting peptidoglycan glycosyltransferases. The uniqueness of moenomycins, in terms of structure and mode of action, and their extreme potency against several Gram-positive pathogens (including vancomycin- and methicillin-resistant cocci), are their advantages as a drug candidate. However, they are not orally bioavailable; in the bloodstream, moenomycins are characterized by an exceedingly long half-life. These shortcomings are thought to be caused by the long C25 lipid chain (moenocinol) of moenomycins. Here we report the generation and initial studies of two ATCC 14672 mutants to access a greater diversity of moenomycins around its lipid chain. The dO5 mutant cannot produce any moenomycins due to the deletion of the moeO5 gene for the first step in moenomycin assembly. The M12 mutant of ATCC 14672 (knockout of the prenyltransferase gene moeN5 ) accumulates moenomycins with a C15 lipid chain instead of moenocinol. We demonstrate and discuss the potential applications of the dO5 and M12 mutants for the discovery of novel moenomycins and their genetic determinants.

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools.

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method.

Pyrazinones are a growing family of microbial NRPS-derived natural products showing interesting biological activities. These compounds are characterized by the presence of either a di- or trisubstituted heterocyclic, nonaromatic 2(1 H)-pyrazinone core in their structure. The most commonly occurring disubstituted pyrazinone natural products are synthesized through a dipeptide intermediate, which is further cyclized to yield the pyrazinone moiety. Trisubstituted pyrazinones are seldom found in natural products, with JBIR56 and JBIR57, isolated from marine Streptomyces , being notable examples. In contrast to the simply organized disubstituted pyrazinones, JBIR56 and JBIR57 are syn-thesized as tetrapeptides with unnatural beta-amino acid residue involved in the for-mation of the pyrazinone moiety. Despite interesting structural features, biosynthetic routes leading to the production of these compounds have not been reported yet. Here we report the discovery of new members of trisubstituted pyrazinone family– tetrapeptides ichizinones A-C in Streptomyces sp. LV45-129. Through sequence analysis and heterologous expression, a biosynthetic gene cluster encoding ichizinone production was identified. Based on gene annotation and sequence homology, a biosynthetic model was suggested. The presented results provide insights into the biosynthesis of rare trisubstituted pyrazinone natural products.

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.

Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the \"capture\" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.