A new synthetic biology system for investigating the biosynthesis of antibiotics and other secondary metabolites in streptomycetes.

We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp*-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors.

Multiple SigB homologues govern the transcription of the ssgBp promoter in the sporulation-specific ssgB gene in Streptomyces coelicolor A3(2).

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues.

Investigating the initial steps of auricin biosynthesis using synthetic biology.

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines.

A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes.

The bacteria of the genus Streptomyces are important producers of a large number of biologically active natural products. Examination of their genomes has revealed great biosynthetic potential for the production of new products, but many of them are silent under laboratory conditions. One of the promising avenues for harnessing this biosynthetic potential is the refactoring and heterologous expression of relevant biosynthetic gene clusters (BGCs) in suitable optimized chassis strains.

A New Family of Transcriptional Regulators Activating Biosynthetic Gene Clusters for Secondary Metabolites.

We previously identified the biosynthetic gene cluster (BGC) in subsp. CCM 3239 (formerly CCM 3239), which is responsible for the production of the unusual angucycline-like antibiotic auricin. Auricin is produced in a narrow interval of the growth phase after entering the stationary phase, after which it is degraded due to its instability at the high pH values reached after the production phase.

The linear plasmid pSA3239 is essential for the replication of the Streptomyces lavendulae subsp. lavendulae CCM 3239 chromosome.

We previously reported the complete genome of Streptomyces lavendulae subsp. lavendulae CCM 3239, containing the linear chromosome and the large linear plasmid pSA3239. Although the chromosome exhibited replication features characteristic for the archetypal end-patching replication, it lacked the tap/tpg gene pair for two proteins essential for this process.

Cross-Recognition of Promoters by the Nine SigB Homologues Present in A3(2).

In contrast to , A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from A3(2).

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties.

The antitumor antibiotic mithramycin: new advanced approaches in modification and production.

The aureolic acid-type polyketide mithramycin (MTM) has a remarkable cytotoxicity against a variety of human tumors and has been used for the treatment of several types of cancer, including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia, and Paget's disease. However, its clinical use is quite limited due to its toxicity. Recently, interest in MTM has been renewed after its identification as a top candidate for the inhibition of the aberrant fusion transcription factor EWS-FLI1, associated with malignant transformation and progression of Ewing sarcoma tumor family.

Pleiotropic anti-anti-sigma factor BldG is phosphorylated by several anti-sigma factor kinases in the process of activating multiple sigma factors in Streptomyces coelicolor A3(2).

The anti-anti-sigma factor BldG has a pleiotropic function in Streptomyces coelicolor A3(2), regulating both morphological and physiological differentiation. Together with the anti-sigma factor UshX, it participates in a partner-switching activation of the sigma factor σ, which has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2).

A Structural Analysis of the Angucycline-Like Antibiotic Auricin from Subsp. CCM 3239 Revealed Its High Similarity to Griseusins.

We previously identified the gene cluster in subsp. CCM 3239 (formerly CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin (). Preliminary characterization of revealed that it possesses an aminodeoxyhexose d-forosamine and is active against Gram-positive bacteria.

Recent achievements in the generation of stable genome alterations/mutations in species of the genus Streptomyces.

The bacteria of the genus Streptomyces are the most valuable source of natural products of industrial and medical importance. A recent explosion of Streptomyces genome sequence data has revealed the enormous genetic potential of new biologically active compounds, although many of them are silent under laboratory conditions. Efficient and stable manipulation of the genome is necessary to induce their production.

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes.

Complete Genome Sequence of subsp. CCM 3239 (Formerly " CCM 3239"), a Producer of the Angucycline-Type Antibiotic Auricin.

subsp. CCM 3239 produces the angucycline antibiotic auricin and was thought to be the type strain of We report the complete genome sequence of this strain, which consists of a linear chromosome and the linear plasmid pSA3239, and demonstrate it to be subsp. .

Correction to: Increased heterologous production of the antitumoral polyketide mithramycin a by engineered Streptomyces lividans TK24 strains.

The original publication contains error error in the Materials and Methods section and in the acknowledgement section.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated.

The σ(F)-specific anti-sigma factor RsfA is one of the protein kinases that phosphorylates the pleiotropic anti-anti-sigma factor BldG in Streptomyces coelicolor A3(2).

The anti-anti-sigma factor BldG has a role in the morphological differentiation and antibiotic production of Streptomyces coelicolor A3(2). Together with the anti-sigma factor UshX it is involved in the "partner-switching"-like activation of the sigma factor σ(H) that has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2).

Strict control of auricin production in Streptomyces aureofaciens CCM 3239 involves a feedback mechanism.

The polyketide gene cluster aur1 is responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is regulated in a complex manner involving several regulators, including a key pathway-specific positive regulator Aur1P that belongs to the family of 'atypical' response regulators. Production of auricin is induced after entry into stationary phase.

Phenotypic analysis of Salmonella enterica serovar Typhimurium rpoE mutants encoding RNA polymerase extracytoplasmic stress response sigma factors σ(E) with altered promoter specificity.

We previously identified mutants in the rpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium) encoding RNA polymerase extracytoplasmic stress response sigma factors σ(E) with altered promoter specificity. The replacement of the conserved R171 residue in the conserved region 4.

Regulation of an alternative sigma factor σI by a partner switching mechanism with an anti-sigma factor PrsI and an anti-anti-sigma factor ArsI in Streptomyces coelicolor A3(2).

Two genes, prsI and arsI, are located divergently next to the sigI gene encoding alternative sigma factor σI of Streptomyces coelicolor A3(2). The similarity of PrsI and ArsI to anti-sigma and anti-anti-sigma factors, respectively, suggests that both putative regulators may be involved in regulation of σI. By using a combination of several approaches including bacterial two-hybrid assays, pull-down assay and visualization of the complex by native polyacrylamide electrophoresis, we demonstrated that PrsI specifically interacts with σI and ArsI.

The dpsA gene of Streptomyces coelicolor: induction of expression from a single promoter in response to environmental stress or during development.

The DpsA protein plays a dual role in Streptomyces coelicolor, both as part of the stress response and contributing to nucleoid condensation during sporulation. Promoter mapping experiments indicated that dpsA is transcribed from a single, sigB-like dependent promoter. Expression studies implicate SigH and SigB as the sigma factors responsible for dpsA expression while the contribution of other SigB-like factors is indirect by means of controlling sigH expression.

The anti-anti-sigma factor BldG is involved in activation of the stress response sigma factor σ(H) in Streptomyces coelicolor A3(2).

The alternative stress response sigma factor σ(H) has a role in regulation of the osmotic stress response and in morphological differentiation in Streptomyces coelicolor A3(2). Its gene, sigH, is located in an operon with the gene that encodes its anti-sigma factor UshX (PrsH). However, no gene with similarity to an anti-anti-sigma factor which may have a role in σ(H) activation by a "partner-switching" mechanism is located in the operon.

Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC.

As free-living non-motile saprophytes, Streptomyces need to adapt to a wide range of environmental conditions and this is reflected by an enormous diversity of regulatory proteins encoded by, for example, the genome of the model streptomycete Streptomyces coelicolor. In this organism, we have identified a new osmoregulation gene, osaC, encoding a member of a novel family of regulatory proteins. Members of the family have a predicted domain composition consisting of an N-terminal kinase domain related to anti-sigma factors, sensory Pas and Gaf domains, and a C-terminal phosphatase domain.

Cascade of sigma factors in streptomycetes: identification of a new extracytoplasmic function sigma factor sigmaJ that is under the control of the stress-response sigma factor sigmaH in Streptomyces coelicolor A3(2).

By using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor A3(2) stress-response sigma factor sigmaH. The promoter directed expression of the sigJ gene encoding an extracytoplasmic function (ECF) sigma factor. S1-nuclease mapping using RNA prepared from E.

Differential production of two antibiotics of Streptomyces coelicolor A3(2), actinorhodin and undecylprodigiosin, upon salt stress conditions.

Production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, is differentially affected in Streptomyces coelicolor A3(2) at high salt concentration, with actinorhodin being inhibited and undecylprodigiosin activated. Analysis of expression of two genes coding for pathway-specific transcriptional regulators of actinorhodin and undecylprodigiosin synthesis, actII-ORF4 and redD, revealed that their expression is similarly differentially affected. Thus, the effect of high salt concentration on actinorhodin and undecylprodigiosin production is mediated at the transcriptional level by the differential expression of genes encoding corresponding pathway-specific transcriptional regulators.