Coordinate regulation of antimycin and candicidin biosynthesis

Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a mere handful of them are expressed in the laboratory, thus poor understanding of how natural products biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species and their regulation is atypical compared to that of most other natural products. Here we demonstrate that antimycin production by Streptomyces albus S4 is regulated by FscRI, a PAS-LuxR-family cluster-situated regulator of the polyene antifungal agent, candicidin. We report that heterologous production of antimycins by Streptomyces coelicolor is dependent on FscRI and show that FscRI activates transcription of key biosynthetic genes. We also demonstrate through ChIP sequencing that FscRI regulation is direct and we provide evidence to suggest that this regulation strategy is conserved and unique to short form antimycin gene clusters. Our study provides direct in vivo evidence for cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products and expands the paradigmatic understanding of the regulation of secondary metabolism

LAL Regulators SCO0877 and SCO7173 as Pleiotropic Modulators of Phosphate Starvation Response and Actinorhodin Biosynthesis in Streptomyces coelicolor

LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era

The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described

Studies on the biosynthesis of antibiotic nystatin with emphasis on regulation

Nystatin is an antifungal polyene macrolide antibiotic produced by Streptomyces noursei, first described in 1950 (Hazen and Brown, 1950). Nystatin is currently being used for treatment of superficial fungal infections, and is considered a medically important drug. However, until recently, nothing was known about the biosynthesis of this antibiotic in S. noursei. This study is the part of the detailed investigation of the genetics and biochemistry of nystatin biosynthesis with emphasis on the regulation. First, the pleiotropic regulatory gene locus from S.noursei capable of enhancing actinorhodin (Act) production in S.lividans was cloned and sequenced. Two genes, designated ssmA and ssmB, have been suggested to be responsible for the phenomenon. Putative product of ssmA showed limited homology to the peptide encoded by afsR2, known as a pleiotropic regulator from S.lividans and S. coelicolor. Recombinant S.lividans strains carrying deletion derivatives of the locus were tested for Act production. The results of these experiments showed that ssmA is required for Act overproduction, while ssmB is possibly involved in the negative regulation of antibiotic production. Further experiments suggested that ssmA is involved in the carbon source-dependent regulation of nystatin production in S.noursei. Next, the entire nystatin biosynthetic gene cluster from S.noursei was cloned and sequenced, putative functions for the biosynthetic genes were implied, and a model for the nystatin biosynthesis was suggested. Six genes encoding PKS type I, genes for posttranslational modifications of nystatin aglycon, efflux of antibiotic out of the cell, and putative clusterspecific regulatory genes have been identified in the cluster. Inactivation of PKS-encoding genes in the cluster resulted in nystatin non-producing mutants, confirming their roles in the biosynthesis of this antibiotic. Genes presumably encoding nystatin efflux pump were studied via gene inactivation and analysis of resulting mutants. It was shown that the efflux is tightly linked to C-10 hydroxylation of the nystatin macrolactone ring. Several genes for post-PKS modifications have been found in the nystatin cluster, among them three genes for synthesis and attachment of mycosamine moiety to the nystatin aglycon. Effect of inactivation of these genes on nystatin biosynthesis was studied. Combined, these results have helped to refine the model of nystatin biosynthesis. The regulatory locus of 6 genes has been found on a right flank of nystatin biosynthetic cluster. Four of them were shown by gene inactivation to be directly involved in the regulation of nystatin biosynthesis. Promoter-probe studies revealed the main targets of regulation in the nystatin gene cluster, and cross-complementation experiments allowed establishing the hierarchy among the regulators. Finally, the model for regulatory cascade was suggested. The results of studies described above provided important information needed for rational engineering of novel polyene macrolides by manipulation of the nystatin biosynthetic genes. Seven analogs of nystatin with altered polyol region and carboxylic group have been obtained and subjected to in vitro antifungal and hemolitic activities tests. It was shown that combinations of several mutations could be beneficial for the activity-toxicity properties of the new compounds. The two most active and less toxic analogs were chosen for in vivo tests in a mouse model, where they proved to be considerably less toxic and at least as active as amphotericin B, the antifungal antibiotic used for treatment of systemic fungal infections. These results indicate that two obtained nystatin analogs can be used for further development into antifungal drugs for human therapy, and that genetic engineering is an effective tool for obtaining new compounds with improved therapeutic properties.PhD i bioteknologiPhD in Biotechnolog

Activation of chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712 by ethanol shock: Insights from the promoter fusion studies

Background Streptomyces venezuelae ATCC 10712 produces antibiotics chloramphenicol (Cml) and jadomycin (Jad) in response to nutrient limitation and ethanol shock (ES), respectively. Biosynthesis of Cml and Jad was shown to be reciprocally regulated via the action of regulatory proteins JadR1 and JadR2 encoded by the jad cluster, and mechanism of such regulation has been characterized. However, detailed analysis of the regulatory mechanism controlling Cml biosynthesis is still lacking. Results In the present study, several promoters from the cml cluster were fused to the reporter gene gusA. Reporter protein activity and Cml production were assayed in the wild-type strain with and without ES, followed by similar experiments with the jadR1 deletion mutant. The latter gene was earlier reported to negatively control Cml biosynthesis, while serving as a positive regulator for the jad cluster. A double deletion mutant deficient in both jadR1 and the cml cluster was also constructed and used in promoter fusion studies. Analyses of the results revealed that ES activates Cml biosynthesis in both wild-type and jadR1 deletion mutant, while Cml production by the latter was ca 80 % lower. Conclusions These results contradict earlier reports regarding the function of JadR1, but correlate well with the reporter activity data for some promoters, while reaction of others to the ES is genotype-dependent. Remarkably, the absence of Cml production in the double mutant has a profound effect on the way certain cml promoters react to ES. The latter suggests direct involvement of Cml in this complex regulatory mechanism

Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.

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

MOESM1 of Activation of chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712 by ethanol shock: insights from the promoter fusion studies

Additional file 1. Supplementary tables (S1–S2) containing information on oligonucleotide primers used for amplification of DNA fragments

Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites

Sun Y-Q, Busche T, Rückert C, et al. Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites. ACS SYNTHETIC BIOLOGY. 2017;6(6):1026-1033.Genome mining of actinomycete bacteria aims at the discovery of novel bioactive secondary metabolites that can be developed into drugs. A new repressor based biosensor to detect activated secondary metabolite biosynthesis gene clusters in Streptomyces was developed. Biosynthetic gene clusters for undecylprodigiosin and coelimycin in the genome of Streptomyces lividans TK24, which encoded TetR-like repressors and appeared to be almost "silent" based on the RNA-seq data, were chosen for the proof-of-principle studies. The bpsA reporter gene for indigoidine synthetase was placed under control of the promotor/operator regions presumed to be controlled by the cluster-associated TetR-like repressors. While the biosensor for undecylprodigiosin turned out to be nonfunctional, the coelimycin biosensor was shown to perform as expected, turning on biosynthesis of indigoidine in response to the concomitant production of coelimycin. The developed reporter system concept can be applied to those cryptic gene clusters that encode metabolite-sensing repressors to speed up discovery of novel bioactive compounds in Streptomyces