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.

Unusual features of the large linear plasmid pSA3239 from Streptomyces aureofaciens CCM 3239.

We previously identified the aur1 gene cluster, responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Pulse-field gel electrophoresis showed a single, 241kb linear plasmid, pSA3239, in this strain, and several approaches confirmed the presence of the aur1 cluster in this plasmid. We report here the nucleotide sequence of this 241,076-bp plasmid.

Characterisation of the genes involved in the biosynthesis and attachment of the aminodeoxysugar D-forosamine in the auricin gene cluster of Streptomyces aureofaciens CCM3239.

We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes.

A γ-butyrolactone autoregulator-receptor system involved in the regulation of auricin production in Streptomyces aureofaciens CCM 3239.

The γ-butyrolactone (GBL) autoregulator-receptor systems play a role in controlling secondary metabolism and/or morphological differentiation in many Streptomyces species. We previously identified the aur1 gene cluster, located on the Streptomyces aureofaciens CCM 3239 large linear plasmid pSA3239, which is responsible for the production of the angucycline antibiotic auricin. Here, we describe the characterisation of two genes, sagA and sagR, encoding GBL autoregulatory signalling homologues, which lie in the upstream part of the aur1 cluster.

Intriguing properties of the angucycline antibiotic auricin and complex regulation of its biosynthesis.

Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin.

A gene determining a new member of the SARP family contributes to transcription of genes for the synthesis of the angucycline polyketide auricin in Streptomyces aureofaciens CCM 3239.

Three regulators, Aur1P, Aur1R and a SARP-family Aur1PR3, have been previously found to control expression of the aur1 cluster for the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Here, we describe an additional regulatory gene, aur1PR4, encoding a homologue from the SARP-family regulators. Its role in auricin regulation was confirmed by its disruption that dramatically affected auricin production.

The gene cluster aur1 for the angucycline antibiotic auricin is located on a large linear plasmid pSA3239 in Streptomyces aureofaciens CCM 3239.

We previously identified a polyketide synthase gene cluster, aur1, responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. A sequence analysis of the aur1 flanking regions revealed the presence of several genes encoding proteins homologous to those for Streptomyces linear plasmid replication, partitioning and telomere-binding. Pulse-field gel electrophoresis detected the single, 240-kb linear plasmid, pSA3239, in S.

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.

Genetic manipulation of pathway regulation for overproduction of angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239.

The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S.