Bogdan Tokovenko

Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes.

We developed a synthetic promoter library for actinomycetes based on the -10 and -35 consensus sequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, in between and downstream of these consensus sequences were randomised using degenerate primers and cloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest synthetic promoter was used in a proof-of-principle approach to achieve the overexpression of a natural type III polyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seq reads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reporter system.

Actinomycetes biosynthetic potential: how to bridge in silico and in vivo?

Actinomycetes genome sequencing and bioinformatic analyses revealed a large number of “cryptic” gene clusters coding for secondary metabolism. These gene clusters have the potential to increase the chemical diversity of natural products. Indeed, reexamination of well-characterized actinomycetes strains revealed a variety of hidden treasures. Growing information about this metabolic diversity has promoted further development of strategies to discover novel biologically active compounds produced by actinomycetes. This new task for actinomycetes genetics requires the development and use of new approaches and tools. Application of synthetic biology approaches led to the development of a set of strategies and tools to satisfy these new requirements. In this review, we discuss strategies and methods to discover small molecules produced by these fascinating bacteria and also discuss a variety of genetic instruments and regulatory elements used to activate secondary metabolism cryptic genes for the overproduction of these metabolites.

Complete genome sequence of Streptomyces fulvissimus.

The complete genome sequence of Streptomyces fulvissimus (DSM 40593), consisting of a linear chromosome with a size of 7.9Mbp, is reported. Preliminary data indicates that the chromosome of S. fulvissimus contains 32 putative gene clusters involved in the biosynthesis of secondary metabolites, two of them showing very high similarity to the valinomycin and nonactin biosynthetic clusters. The availability of genome sequence of S. fulvissimus will contribute to the evaluation of the full biosynthetical potential of streptomycetes.

Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of 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.

Insights into the Pamamycin Biosynthesis

Pamamycins are macrodiolides of polyketide origin with antibacterial activities. Their biosynthesis has been proposed to utilize succinate as a building block. However, the mechanism of succinate incorporation into a polyketide was unclear. Here, we report identification of a pamamycin biosynthesis gene cluster by aligning genomes of two pamamycin-producing strains. This unique cluster contains polyketide synthase (PKS) genes encoding seven discrete ketosynthase (KS) enzymes and one acyl-carrier protein (ACP)-encoding gene. A cosmid containing the entire set of genes required for pamamycin biosynthesis was successfully expressed in a heterologous host. Genetic and biochemical studies allowed complete delineation of pamamycin biosynthesis. The pathway proceeds through 3-oxoadipyl-CoA, a key intermediate in the primary metabolism of the degradation of aromatic compounds. 3-Oxoadipyl-CoA could be used as an extender unit in polyketide assembly to facilitate the incorporation of succinate.

New natural products identified by combined genomics-metabolomics profiling of marine Streptomyces sp. MP131-18

Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family – lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.

New Simocyclinones: Surprising Evolutionary and Biosynthetic Insights

Simocyclinone D8 (1, SD8) has attracted attention due to its highly complex hybrid structure and the unusual way it inhibits bacterial DNA gyrase by preventing DNA binding to the enzyme. Although a hypothesis explaining simocyclinone biosynthesis has been previously proposed, little was proven in vivo due to the genetic inaccessibility of the producer strain. Herein, we report discovery of three new D-type simocyclinones (D9, D10, and D11) produced by Kitasatospora sp. and Streptomyces sp. NRRL B-24484, as well as the identification and annotation of their biosynthetic gene clusters. Unexpectedly, the arrangement of the newly discovered biosynthetic gene clusters is starkly different from the previously published one, despite the nearly identical structures of D8 and D9 simocyclinones. The gene inactivation and expression studies have disproven the role of a modular polyketide synthase (PKS) system in the assembly of the linear dicarboxylic acid. Instead, the new stand-alone ketosynthase genes were shown to be involved in the biosynthesis of the tetraene chain. Additionally, we identified the gene responsible for the conversion of simocyclinone D9 (2, SD9) into D8.

Identification of butenolide regulatory system controlling secondary metabolism in Streptomyces albus J1074

A large majority of genome-encrypted chemical diversity in actinobacteria remains to be discovered, which is related to the low level of secondary metabolism genes expression. Here, we report the application of a reporter-guided screening strategy to activate cryptic polycyclic tetramate macrolactam gene clusters in Streptomyces albus J1074. The analysis of the S. albus transcriptome revealed an overall low level of secondary metabolism genes transcription. Combined with transposon mutagenesis, reporter-guided screening resulted in the selection of two S. albus strains with altered secondary metabolites production. Transposon insertion in the most prominent strain, S. albus ATGSal2P2::TN14, was mapped to the XNR_3174 gene encoding an unclassified transcriptional regulator. The mutant strain was found to produce the avenolide-like compound butenolide 4. The deletion of the gene encoding a putative acyl-CoA oxidase, an orthologue of the Streptomyces avermitilis avenolide biosynthesis enzyme, in the S. albus XNR_3174 mutant caused silencing of secondary metabolism. The homologues of XNR_3174 and the butenolide biosynthesis genes were found in the genomes of multiple Streptomyces species. This result leads us to believe that the discovered regulatory elements comprise a new condition-dependent system that controls secondary metabolism in actinobacteria and can be manipulated to activate cryptic biosynthetic pathways.

The isolation and characterization of actinobacteria from dominant benthic macroinvertebrates endemic to Lake Baikal

The high demand for new antibacterials fosters the isolation of new biologically active compounds producing actinobacteria. Here, we report the isolation and initial characterization of cultured actinobacteria from dominant benthic organisms’ communities of Lake Baikal. Twenty-five distinct strains were obtained from 5 species of Baikal endemic macroinvertebrates of amphipods, freshwater sponges, turbellaria worms, and insects (caddisfly larvae). The 16S ribosomal RNA (rRNA)-based phylogenic analysis of obtained strains showed their affiliation to Streptomyces, Nocardia, Pseudonocardia, Micromonospora, Aeromicrobium, and Agromyces genera, revealing the diversity of actinobacteria associated with the benthic organisms of Lake Baikal. The biological activity assays showed that 24 out of 25 strains are producing compounds active against at least one of the test cultures used, including Gram-negative bacteria and Candida albicans. Complete dereplication of secondary metabolite profiles of two isolated strains led to identification of only few known compounds, while the majority of detected metabolites are not listed in existing antibiotic databases.

Generation of new compounds through unbalanced transcription of landomycin A cluster

The biosynthetically well-studied landomycin A cluster has been used to demonstrate the unbalancing of gene transcription as an efficient method for the generation of new compounds. Landomycin A structural genes were decoupled from the native regulators LanI and LanK and placed under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. In contrast to their native quantitative and temporal regulation, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article.