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.

Transcription from the chiC promoter, directing expression of the chitinase gene, chiC, in Streptomyces coelicolor, was analyzed using xylE reporter gene and high-resolution S1-nuclease mapping. The transcription from the chiC promoter was induced by chitin, and this induction was dramatically reduced in the S. coelicolor chiR-disrupted strain. This indicated a dependence of chiC expression upon the chiR gene encoding a response regulator protein. To investigate this relationship, the S. coelicolor ChiR was overproduced using Escherichia coli T7 RNA polymerase expression system. However, gel mobility shift-assay with such a purified ChiR showed no binding in the chiC promoter region, which indicates a lack of specific phosphorylation of E. coli overproduced ChiR that is necessary for DNA-binding activity of response regulators.

A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strain Streptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.