Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

A gene, aur1P, encoding a protein similar to the response regulators of bacterial two-component signal transduction systems, was identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. Expression of the gene was directed by a single promoter, aur1Pp, which was transcribed at low levels during the exponential phase and induced just before the stationary phase. A divergently transcribed gene, aur1R, has been identified upstream of aur1P, encoding a protein homologous to transcriptional repressors of the TetR family. The aur1P gene was disrupted in the S. aureofaciens CCM 3239 chromosome by homologous recombination. The mutation in the aur1P gene had no effect on growth and differentiation. However, biochromatographic analysis of culture extracts from the S. aureofaciens aur1P-disrupted strain revealed that auricin was not produced in the mutant. This indicated that aur1P is essential for auricin production. Transcription from the previously characterized aur1Ap promoter, directing expression of the first gene, aur1A, in the auricin gene cluster, was dramatically decreased in the S. aureofaciens CCM 3239 aur1P mutant strain. Moreover, the Aur1P protein, overproduced in Escherichia coli, was shown to bind specifically upstream of the aur1Ap promoter region. The results indicated that the Aur1P regulator activates expression of the auricin biosynthesis genes.

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. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

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. However, auricin was produced in only a short time interval of several hours. We found that the decrease of auricin production was due to a strict regulation of auricin biosynthetic genes at the transcriptional level by a feedback mechanism; auricin and/or its intermediate(s) inhibited binding of Aur1P to its cognate biosynthetic promoter aur1Ap and consequently stopped its activation. In addition, we also determined that synthesised auricin is unstable during growth of S. aureofaciens CCM3239 in the production medium even though purified auricin is stable for days in various organic solvents. The critical parameter affecting its stability was pH. Auricin is stable at acid pH and unstable at neutral and alkaline pH. The drop in auricin concentration was due to an increase of pH shortly after induction of auricin production during cultivation of S. aureofaciens CCM3239.

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. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.

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. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.