Elke Brötz

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.

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.

Phenelfamycins G and H, new elfamycin-type antibiotics produced by Streptomyces albospinus Acta 3619

Phenelfamycins G and H are new members of the family of elfamycin antibiotics with the basic structure of phenelfamycins E and F, respectively, which are also well known as ganefromycins α and β. Phenelfamycins G and H differ from phenelfamycins E and F by an additional hydroxy group at position C-30, which is not described so far for any of the elfamycin-type antibiotics. The actinomycete strain that produced phenelfamycins G and H was identified to be Streptomyces albospinus based on its 16S rRNA gene sequence. Phenelfamycins G and H exhibit a narrow antibacterial spectrum with a pronounced inhibitory activity against Propionibacterium acnes.

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.

Insights into the Bioactivity of Mensacarcin and Epoxide Formation by MsnO8

Mensacarcin, a potential antitumour drug, is produced by Streptomyces bottropensis. The structure consists of a three‐membered ring system with many oxygen atoms. Of vital importance in this context is an epoxy moiety in the side chain of mensacarcin. Our studies with different mensacarcin derivatives have demonstrated that this epoxy group is primarily responsible for the cytotoxic effect of mensacarcin. In order to obtain further information about this epoxy moiety, inactivation experiments in the gene cluster were carried out to identify the epoxy‐forming enzyme. Therefore the cosmid cos2, which covers almost the complete type II polyketide synthase (PKS) gene cluster, was heterologously expressed in Streptomyces albus. This led to production of didesmethylmensacarcin, due to the fact that methyltransferase genes are missing in the cosmid. Further gene inactivation experiments on this cosmid showed that MsnO8, a luciferase‐like monooxygenase, introduces the epoxy group at the end of the biosynthesis of mensacarcin. In addition, the protein MsnO8 was purified, and its crystal structure was determined to a resolution of 1.80 Å.

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.

Functional Characterization of Different ORFs Including Luciferase‐Like Monooxygenase Genes from the Mensacarcin Gene Cluster

The biologically active compound mensacarcin is produced by Streptomyces bottropensis. The cosmid cos2 contains a large part of the mensacarcin biosynthesis gene cluster. Heterologous expression of this cosmid in Streptomyces albus J1074 led to the production of the intermediate didesmethylmensacarcin (DDMM). In order to gain more insights into the biosynthesis, gene inactivation experiments were carried out by λ‐Red/ET‐mediated recombination, and the deletion mutants were introduced into the host S. albus. In total, 23 genes were inactivated. Analysis of the metabolic profiles of the mutant strains showed the complete collapse of DDMM biosynthesis, but upon overexpression of the SARP regulatory gene msnR1 in each mutant new intermediates were detected. The compounds were isolated, and their structures were elucidated. Based on the results the specific functions of several enzymes were determined, and a pathway for mensacarcin biosynthesis is proposed.

Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

ABSTRACT Saccharothrix espanaensis is a member of the order Actinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase from Saccharothrix espanaensis which is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.