Deletions of conserved extracytoplasmic function sigma factors-encoding genes in Streptomyces have a major impact on secondary metabolism.

BACKGROUND: Ethanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function. RESULTS: To establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism. CONCLUSIONS: These findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.

Targeted Metabolomics and High-Throughput RNA Sequencing-Based Transcriptomics Reveal Massive Changes in the Streptomyces venezuelae NRRL B-65442 Metabolism Caused by Ethanol Shock.

The species Streptomyces venezuelae is represented by several distinct strains with variable abilities to biosynthesize structurally diverse secondary metabolites. In this work, we examined the effect of ethanol shock on the transcriptome and metabolome of Streptomyces venezuelae NRRL B-65442 using high-throughput RNA sequencing (RNA-seq) and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ethanol shock caused massive changes in the gene expression profile, differentially affecting genes for secondary metabolite biosynthesis and central metabolic pathways. Most of the data from the transcriptome analysis correlated well with the metabolome changes, including the overproduction of jadomycin congeners and a downshift in the production of desferrioxamines, legonoxamine, foroxymithin, and a small cryptic ribosomally synthesized peptide. Some of the metabolome changes, such as the overproduction of chloramphenicol, could not be explained by overexpression of the cognate biosynthetic genes but correlated with the expression profiles of genes for precursor biosynthesis. Changes in the transcriptome were also observed for several genes known to play a role in stress response in other bacteria and included at least 10 extracytoplasmic function σ factors. This study provides important new insights into the stress response in antibiotic-producing bacteria and will help to understand the complex mechanisms behind the environmental factor-induced regulation of secondary metabolite biosynthesis. IMPORTANCE Streptomyces spp. are filamentous Gram-positive bacteria known as versatile producers of secondary metabolites, of which some have been developed into human medicines against infections and cancer. The genomes of these bacteria harbor dozens of gene clusters governing the biosynthesis of secondary metabolites (BGCs), of which most are not expressed under laboratory conditions. Detailed knowledge of the complex regulation of BGC expression is still lacking, although certain growth conditions are known to trigger the production of previously undetected secondary metabolites. In this work, we investigated the effect of ethanol shock on the production of secondary metabolites by Streptomyces venezuelae and correlated these findings with the expression of cognate BGCs and primary metabolic pathways involved in the generation of cofactors and precursors. The findings of this study set the stage for the rational manipulation of bacterial genomes aimed at enhanced production of industrially important bioactive natural products.

Coupling of the engineered DNA "mutator" to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces.

DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these 'mutators' was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10-5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

Noursamycins, Chlorinated Cyclohexapeptides Identified from Molecular Networking of Streptomyces noursei NTR-SR4.

The noursamycins A-F are chlorinated cyclic hexapeptides, which were identified and isolated from the strain Streptomyces noursei NTR-SR4 overexpressing a LuxR-like transcriptional activator. The molecules were structurally characterized by mass spectrometric analyses and 1D and 2D NMR spectroscopic techniques. The enzymatic machinery involved in the biosynthesis of these peptides is represented by a modular nonribosomal peptide synthetase (NRPS), and the corresponding gene cluster was identified in the S. noursei genome. The latter suggested the biosynthetic pathway for the noursamycins. Spectral networking analysis uncovered noursamycin derivatives that were later found to result from a relaxed substrate specificity of the A3 and A4 adenylation domains of the NRPS. The stereochemistry of the amino acid constituents of the noursamycins was resolved by chemical derivatization, subsequent enantiomer analytics by GC-EIMS, and in silico data analyses. Noursamycins A and B exhibited antibacterial activity against Gram-positive and Gram-negative bacteria, while no apparent cytotoxicity was observed.

Novel bioactive natural products from bacteria via bioprospecting, genome mining and metabolic engineering.

For over seven decades, bacteria served as a valuable source of bioactive natural products some of which were eventually developed into drugs to treat infections, cancer and immune system-related diseases. Traditionally, novel compounds produced by bacteria were discovered via conventional bioprospecting based on isolation of potential producers and screening their extracts in a variety of bioassays. Over time, most of the natural products identifiable by this approach were discovered, and the pipeline for new drugs based on bacterially produced metabolites started to run dry. This mini-review highlights recent developments in bacterial bioprospecting for novel compounds that are based on several out-of-the-box approaches, including the following: (i) targeting bacterial species previously unknown to produce any bioactive natural products, (ii) exploring non-traditional environmental niches and methods for isolation of bacteria and (iii) various types of 'genome mining' aimed at unravelling genetic potential of bacteria to produce secondary metabolites. All these approaches have already yielded a number of novel bioactive compounds and, if used wisely, will soon revitalize drug discovery pipeline based on bacterial natural products.

An orthogonal system for heterologous expression of actinobacterial lasso peptides in Streptomyces hosts.

Lasso peptides are ribosomally synthesized and post-translationally modified peptides produced by bacteria. They are characterized by an unusual lariat-knot structure. Targeted genome scanning revealed a wide diversity of lasso peptides encoded in actinobacterial genomes, but cloning and heterologous expression of these clusters turned out to be problematic. To circumvent this, we developed an orthogonal expression system for heterologous production of actinobacterial lasso peptides in Streptomyces hosts based on a newly-identified regulatory circuit from Actinoalloteichus fjordicus. Six lasso peptide gene clusters, mainly originating from marine Actinobacteria, were chosen for proof-of-concept studies. By varying the Streptomyces expression hosts and a small set of culture conditions, three new lasso peptides were successfully produced and characterized by tandem MS. The newly developed expression system thus sets the stage to uncover and bioengineer the chemo-diversity of actinobacterial lasso peptides. Moreover, our data provide some considerations for future bioprospecting efforts for such peptides.

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

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.

New Deferoxamine Glycoconjugates Produced upon Overexpression of Pathway-Specific Regulatory Gene in the Marine Sponge-Derived Streptomyces albus PVA94-07.

Activation of silent biosynthetic gene clusters in Streptomyces bacteria via overexpression of cluster-specific regulatory genes is a promising strategy for the discovery of novel bioactive secondary metabolites. This approach was used in an attempt to activate a cryptic gene cluster in a marine sponge-derived Streptomyces albus PVA94-07 presumably governing the biosynthesis of peptide-based secondary metabolites. While no new peptide-based metabolites were detected in the recombinant strain, it was shown to produce at least four new analogues of deferoxamine with additional acyl and sugar moieties, for which chemical structures were fully elucidated. Biological activity tests of two of the new deferoxamine analogues revealed weak activity against Escherichia coli. The gene knockout experiment in the gene cluster targeted for activation, as well as overexpression of certain genes from this cluster did not have an effect on the production of these compounds by the strain overexpressing the regulator. It seems plausible that the production of such compounds is a response to stress imposed by the production of an as-yet unidentified metabolite specified by the cryptic cluster.

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.

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.

Engineering terpene biosynthesis in Streptomyces for production of the advanced biofuel precursor bisabolene.

The past decade has witnessed a large influx of research toward the creation of sustainable, biologically derived fuels. While significant effort has been exerted to improve production capacity in common hosts, such as Escherichia coli or Saccharomyces cerevisiae, studies concerning alternate microbes comparatively lag. In an effort to expand the breadth of characterized hosts for fuel production, we map the terpene biosynthetic pathway in a model actinobacterium, Streptomyces venezuelae, and further alter secondary metabolism to afford the advanced biofuel precursor bisabolene. Leveraging information gained from study of the native isoprenoid pathway, we were able to increase bisabolene titer nearly 5-fold over the base production strain, more than 2 orders of magnitude greater than the combined terpene yield in the wild-type host. We also explored production on carbon sources of varying complexity to, notably, define this host as one able to perform consolidated bioprocessing.

Genomics of sponge-associated Streptomyces spp. closely related to Streptomyces albus J1074: insights into marine adaptation and secondary metabolite biosynthesis potential.

A total of 74 actinomycete isolates were cultivated from two marine sponges, Geodia barretti and Phakellia ventilabrum collected at the same spot at the bottom of the Trondheim fjord (Norway). Phylogenetic analyses of sponge-associated actinomycetes based on the 16S rRNA gene sequences demonstrated the presence of species belonging to the genera Streptomyces, Nocardiopsis, Rhodococcus, Pseudonocardia and Micromonospora. Most isolates required sea water for growth, suggesting them being adapted to the marine environment. Phylogenetic analysis of Streptomyces spp. revealed two isolates that originated from different sponges and had 99.7% identity in their 16S rRNA gene sequences, indicating that they represent very closely related strains. Sequencing, annotation, and analyses of the genomes of these Streptomyces isolates demonstrated that they are sister organisms closely related to terrestrial Streptomyces albus J1074. Unlike S. albus J1074, the two sponge streptomycetes grew and differentiated faster on the medium containing sea water. Comparative genomics revealed several genes presumably responsible for partial marine adaptation of these isolates. Genome mining targeted to secondary metabolite biosynthesis gene clusters identified several of those, which were not present in S. albus J1074, and likely to have been retained from a common ancestor, or acquired from other actinomycetes. Certain genes and gene clusters were shown to be differentially acquired or lost, supporting the hypothesis of divergent evolution of the two Streptomyces species in different sponge hosts.

Genome-based bioprospecting of microbes for new therapeutics.

Bioprospecting of natural sources for new medicines has a long and successful history, exemplified by the fact that over 50% of all drugs currently on the market are either derived from or inspired by natural products. However, development of new natural product-based therapeutics has been on the decline over the past 20 years, mainly owing to frequent re-discovery of already known compounds coupled with high costs for screening, characterization and development. With the onset of the genomic era allowing rapid sequencing and analysis of bacterial and fungal genomes, it became evident that these organisms possess 'hidden treasures' in the form of gene clusters potentially governing biosynthesis of novel biologically active compounds. This review highlights current progress in mining for and expression of these gene clusters, which may revolutionize the drug discovery pipelines in the near future.

New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei.

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

Improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces noursei.

Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

Analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455.

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

Nystatin biosynthesis and transport: nysH and nysG genes encoding a putative ABC transporter system in Streptomyces noursei ATCC 11455 are required for efficient conversion of 10-deoxynystatin to nystatin.

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

An unexpected role for the putative 4'-phosphopantetheinyl transferase-encoding gene nysF in the regulation of nystatin biosynthesis in Streptomyces noursei ATCC 11455.

The nysF gene encoding a putative 4'-phosphopantetheinyl transferase (PPTase) is located at the 5' border of the nystatin biosynthesis gene cluster in Streptomyces noursei. PPTases carry out post-translational modification of the acyl carrier protein domains on the polyketide synthases (PKS) required for their full functionality, and hence NysF was assumed to be involved in similar modification of the nystatin PKS. At the same time, DNA sequence analysis of the genomic region adjacent to the nysF gene revealed a gene cluster for a putative lantibiotic biosynthesis. This finding created some uncertainty regarding which gene cluster nysF functionally belongs to. To resolve this ambiguity, nysF was inactivated by both insertion of a kanamycin (Km) resistance marker into its coding region, and by in-frame deletion. Surprisingly, the nystatin production in both the nysF::Km(R) and DeltanysF mutants increased by ca. 60% compared to the wild-type, suggesting a negative role of nysF in the nystatin biosynthesis. The expression of xylE reporter gene under control of different promoters from the nystatin gene cluster in the DeltanysF mutant was studied. The data obtained clearly show enhanced expression of xylE from the promoters of several structural and regulatory genes in the DeltanysF mutant, implying that NysF negatively regulates the nystatin biosynthesis.

In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis.

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.