Implication of orphan histidine kinase (OhkAsp) in biosynthesis of doxorubicin and daunorubicin in Streptomyces peucetius ATCC 27952.

The orphan histidine kinase (HK) from Streptomyces peucetius ATCC 27952 (ohkAsp) was found to be implicated in the regulation of doxorubicin (DOX)/daunorubicin (DNR) biosynthesis, self-defense and developmental attributes. OhkAsp is a homolog of OhkA from Streptomyces coelicolor and Streptomyces avermitilis (with 73 and 75% identity). As in its homologs, S. peucetius mutant with deletion of ohkAsp was found to enhance metabolite biosynthesis and impaired the morphological differentiation. But, unlike its homologs from Streptomyces coelicolor and Streptomyces avermitilis, differential enhancement in level of secondary metabolite production was found in overexpression mutants apart from deletion mutant. The deflection in characteristics of OhkA in its homologue from S. peucetius ATCC 27952, and its imminent implications was monitered by making various mutants with differential expression level of ohkAsp. The variations were observed in the morphology of mutants, transcriptional level of effectors and regulators of DOX/DNR biosynthesis pathway, DOX/DNR precursor pool and biomass accumulation. Based on comparisons of domain arrangements among its homologs, Low Complexity Region (LCR) present on the OhkAsp was the only domain that stood out. Further, the LCR on OhkAsp was found to be overlapping with a putative receiver domain responsible for interaction with response regulator. The imminent implications of differential expression level of ohkAsp on: regulation and biosynthesis of DOX/DNR, morphological differentiation, DOX/DNR precursor pool and biomass accumulation were explored in this study.

Complete genome sequence of Streptomyces peucetius ATCC 27952, the producer of anticancer anthracyclines and diverse secondary metabolites.

Streptomyces peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. peucetius ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.

DnrI of Streptomyces peucetius binds to the resistance genes, drrAB and drrC but is activated by daunorubicin.

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952.

The dnrO gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of Streptomyces peucetius ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. peucetius was found to contain no functional copy of bldA-tRNA while a detailed examination of dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA-tRNA. Therefore, for evaluating the role of dnrO in DNR production, multiple engineered strains of S. peucetius were generated by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, the effects of heterologously expressed bldA and overexpressed dnrO were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of dnrO and master regulator dnrI, was found to be elevated in bldA containing strain in comparison to dnrO overexpressed strain. The bldA containing strain produces 45.7% higher DNR than bldA deficient wild type strain from culture broth with OD600 of 1.45 at 72h. Heterologous expression of bldA-tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.

[Influence of perfluorodecalin on growth of actinomycetes and intensification of Streptomycin and daunorubicin production by the genus Streptomyces kind bacteria in submerged culture].

Addition of perfluorodecalin with gas-transporting function to the liquid medium during submerged cultivation of actinomycetes of the genus Streptomyces resulted in higher intensity and level of the biomass synthesis and increased production of streptomycin and daunorubicin. Addition of perfluorodecalin to the medium provided a 2.0-2.3-fold surpass of the maximum antibiotic production (achieved by the 120th-144th hours of the culture growth) vs. the antibiotic accumulation peaks in the control.

Regulation of daunorubicin biosynthesis in Streptomyces peucetius - feed forward and feedback transcriptional control.

Streptomyces are a major group of soil bacteria that produce wide range of bioactive compounds including antibiotics. Daunorubicin is a chemotherapeutic agent for treatment of certain types of cancer, which is produced as a secondary metabolite by S. peucetius. Owing to the significance of this drug in treating cancer, understanding the molecular mechanism of its biosynthesis will assist in the genetic manipulation of this strain for better drug yields. Additionally, the knowledge can also be applied to design hybrid antibiotics that can be made in vivo by transferring genes from one Streptomyces species to another. Biosynthesis of daunorubicin in S. peucetius is accomplished by the function of 30 enzyme-coding genes in a sequential and coordinated fashion. In addition to these enzymes, three transcriptional regulators DnrO, DnrN and DnrI regulate this multi-step process by forming a coherent feed forward loop regulatory circuit, consequently controlling the entire enzyme coding genes. Since daunorubicin is a DNA intercalating drug, maintaining an optimal intracellular drug concentration is pivotal to prevent self-toxicity. Commencement of daunorubicin biosynthesis also activates the feedback mechanisms mediated by the metabolite. At exceeding intracellular concentrations, daunorubicin intercalates into DNA sequences and impedes the binding of these transcription factors. This feedback repression is relieved by a group of self-resistance genes, which concurrently efflux the excess intracellular daunorubicin. This review will discuss the mechanistic role of each transcription factor and their interplay in initiating and maintaining the biosynthesis of daunorubicin in S. peucetius.

Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952.

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.

Improvement of antibiotic productivity by knock-out of dauW in Streptomyces coeruleobidus.

Daunorubicin (DNR) is an important anthracycline antibiotic. Its biosynthesis pathway has been well understood, however, the regulation of DNR biosynthesis needs further investigations. An ORF cloned between drrB and dnrX from the genome of a DNR producer, Streptomyces coeruleobidus DM, was named dauW and designated as an orthologous gene with dnrW and drrD. Several plasmids were constructed for over-expression and/or disruption of dauW in DM. Complete disruption of dauW can significantly increase the yield of DNR. We also found that the transcription level of dnrI, a major regulatory protein in the biosynthesis of DNR, and the self-resistance level were improved in dauW knock-out mutant. These results suggested that dauW may be a down-regulatory gene for DNR biosynthesis. Antibiotics productivity in S. coeruleobidus could be improved via regulation of the transcription of dnrI, a SARP regulator. The production of DNR in a high-producer and the yield of epi-DNR in an engineering strain were also increased by disruption of dauW.

Daunorubicin efflux in Streptomyces peucetius modulates biosynthesis by feedback regulation.

Streptomyces peucetius self-resistance genes drrA and drrB encode membrane-associated proteins that function like an ABC transporter for the efflux of daunorubicin and to maintain a constant subinhibitory physiological concentration of the drug within the cell. In this study, the drrA and drrB operons were disrupted for investigating drug production, self-resistance and regulation. The drrA-drrB null mutant was highly sensitive to daunorubicin. A 10-fold decrease in drug production was observed in the null mutant compared with the wild-type strain. We propose that the absence of a drug-specific efflux pump increases the intracellular concentration of daunorubicin, which is sensed by the organism to turn down drug production. Quantitative real-time PCR analysis of the mutant showed a drastic reduction in the expression of the key regulator dnrI and polyketide synthase gene dpsA. However, the expression of regulatory genes dnrO and dnrN was increased. Feedback regulation based on the intracellular daunorubicin concentration is discussed.

Isolation and genetic manipulation of the antibiotic down-regulatory gene, wblA ortholog for doxorubicin-producing Streptomyces strain improvement.

Cross-genome comparative transcriptome analyses were previously conducted using the sequenced Streptomyces coelicolor genome microarrays to understand the genetic nature of doxorubicin (DXR) and daunorubicin (DNR) overproducing industrial mutant (OIM) of Streptomyces peucetius. In this previous work, a whiB-like putative transcription factor (wblA ( sco )) was identified as a global antibiotic down-regulator in S. coelicolor (Kang et al., J Bacteriol 189:4315-4319, 2007). In this study, a total genomic DNA library of a DXR/DNR-overproducing S. peucetius OIM was constructed and screened using wblA ( sco ) as a probe, resulting in the isolation of a wblA ortholog (wblA ( spe )) that had 95% amino acid identity to wblA ( sco ). Gene disruption of wblA ( spe ) from the S. peucetius OIM resulted in an approximately 70% increase in DXR/DNR productivity, implying that the DXR/DNR production in the S. peucetius OIM could be further improved via comparative transcriptomics-guided target gene manipulation. Furthermore, several putative wblA ( spe ) -dependent genes were also identified using S. coelicolor interspecies DNA microarray analysis between the S. peucetius OIM and wblA ( spe )-disrupted S. peucetius OIM. Among the genes whose expressions were significantly stimulated in the absence of wblA ( spe ), the overexpression of a conserved hypothetical protein (SCO4967) further stimulated the total production of DXR/DNR/akavinone by 1.3-fold in the wblA ( spe )-disrupted S. peucetius OIM, implying that the sequential genetic manipulation of target genes identified from interspecies comparative microarray analysis could provide an efficient and rational strategy for Streptomyces strain improvement.

A novel protein that binds to dnrN-dnrO intergenic region of Streptomyces peucetius purified by DNA affinity capture has dihydrolipoamide dehydrogenase activity.

An antitumour chemotherapeutic, daunorubicin (DNR), produced by Streptomyces peucetius exhibits cytotoxic activity through topoisomerase-mediated interaction with DNA, thereby inhibiting DNA replication and repair and RNA and protein synthesis. It is synthesized by the type II polyketide pathway. Understanding molecular mechanisms that drive expression of antibiotic biosynthetic genes in response to diverse signals and chemical inducers is of considerable interest. Intergenic DNA between regulatory genes dnrN and dnrO of DNR biosynthesis pathway in S. peucetius has a promoter for transcription of dnrN in one strand and three promoters in the opposite strand for dnrO. Studies have shown that DnrO binds to a specific sequence in this region to activate transcription of dnrN. In the present study, using biotinylated intergenic DNA in combination with streptavidin magnetic beads, we have purified a protein that binds to this target sequence. The protein has been characterized by nano LC ESI MS/MS mass spectrometry. Sequence similarity searches for effective identification of protein by genome databases comparisons led to identification of a sequence-specific DNA binding protein that exhibits dihydrolipoamide dehydrogenase (DLDH) activity suggesting that this protein may be involved in regulation of DNR biosynthesis.

Feedback regulation of doxorubicin biosynthesis in Streptomyces peucetius.

DnrO, one of three DNA binding regulatory proteins involved in daunorubicin biosynthesis in Streptomyces peucetius, has been purified as a maltose-binding protein-DnrO (MBP-DnrO) fusion protein. Gel mobility shift assays showed that it specifically bound to a DNA fragment containing both dnrN and dnrO promoters. In the presence of some low-molecular-weight compounds from the daunorubicin biosynthetic pathway, the DNA binding ability of MBP-DnrO was affected. Melanin production assays showed that both DnrO and DnrN were required for the increased activity of the dnrI promoter. Rhodomycin D (RHOD), one of the intermediates in the DNR and DXR biosynthetic pathways, had a positive effect on dnrI promoter activity only in the presence of both DnrO and DnrN proteins. The promoter activity of dnrO gene decreased in the presence of the DnrO protein, suggesting that dnrO gene was autoregulated. Repression could be relieved when RHOD was present in the culture, indicating that RHOD might directly interact with the DnrO protein.

Engineered biosynthesis of aklanonic acid analogues.

Aklanonic acid, an anthraquinone natural product, is a common advanced intermediate in the biosynthesis of several antitumor polyketide antibiotics, including doxorubicin and aclacinomycin A. Intensive semisynthetic and biosynthetic efforts have been directed toward developing improved analogues of these clinically important compounds. The primer unit of such polyfunctional aromatic polyketides is an attractive site for introducing novel chemical functionality, and attempts have been made to modify the primer unit by precursor-directed biosynthesis or protein engineering of the polyketide synthase (PKS). We have previously demonstrated the feasibility of engineering bimodular aromatic PKSs capable of synthesizing unnatural hexaketides and octaketides. In this report, we extend this ability by preparing analogues of aklanonic acid, a decaketide, and its methyl ester. For example, by recombining the R1128 initiation module with the dodecaketide-specific pradimicin PKS, the isobutyryl-primed analogue of aklanonic acid (YT296b, 10) and its methyl ester (YT299b, 12) were prepared. In contrast, elongation modules from dodecaketide-specific spore pigment PKSs were unable to interact with the R1128 initiation module. Thus, in addition to revealing a practical route to new anthracycline antibiotics, we also observed a fundamental incompatibility between antibiotic and spore pigment biosynthesis in the actinomycetes bacteria.

Crystal structure of a ternary complex of DnrK, a methyltransferase in daunorubicin biosynthesis, with bound products.

One of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-L-methionine-dependent carminomycin 4-O-methyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-epsilon-rhodomycin T has been determined to a 2.35-angstroms resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an SN2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement.

[Effect of perfluoroorganic compounds on growth of streptomycetes and biosynthesis of antibiotic daunorubicin]. / Vliianie perftoroorganicheskikh soedinenii na rost streptomitsetov i biosintez imi antibiotika daunorubitsina.

The use of perfluoroorganic compounds, i.e. perfluorodecalin and "Perftoran", a blood substitute with the gas transfer function, known as "blue blood" (containing 3 vol. % of perfluoromethylcyclohexylpiperidin and 7 vol. % of perfluorodecalin as emulsion with the average particle size of 0.07 microns) in biotechnology for intensification of the Streptomyces purpurogeniscleroticus growth under submerged conditions was shown promising for the first time. An increase of the biomass output and higher yields of daunorubicin were also demonstrated.

Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon.

[Characteristics of mutants of Streptomyces peucetius subsp. caesius ATCC 27952 resistant to anthracycline antibiotics]. / Kharakterystyka mutantiv Streptomyces peucetius subsp. caesius ATCC 27952, stiikykh do antratsyklinovykh antybiotykiv.

Daunorubicine (DNR) and doxorubicine (DXR) resistance to anthracycline antibiotics and antibiotical activity of the strain Streptomyces peucetius subsp. caesius ATCC 27952-2 and its daunorubicine- and doxorubicine-resistant (DNRr and DXRr) mutants have been studied. It has been found that strain S. peucetius subsp. caesius ATCC 27952-2 is much more resistant to DXR than to DNR. It has been shown that frequency of appearance of spontaneous mutants of the strain S. peucetius subsp. caesius ATCC 27952-2, resistant to DNR (10-30 micrograms/ml) makes 1.1 x 10(-6)-1.0 x 10(-8) and DXR (10-30 micrograms/ml) 7.7 x 10(-4)-3.0 x 10(-4). Treatment of spores of the initial strain by N-methyl-N'-nitro-N-nitrosoguanidin resulted in the 60-700-fold increase of arising frequency of DNRr-mutants while frequency of arising of DXRr-mutants did not change essentially. The majority of DNRr-mutants (76.9%) were characterised by high resistance to DXR, while there are such mutants among DXRr-mutants which possess the cross-resistance to DNR (below 30%). The mean value of antibiotic activity was the highest among DXRr-mutants induced by NG and sampled in the medium with 30 micrograms/ml of DXR. The majority of the tested DNRr- and DXRr-mutants (13 of 15) synthesized 1.8-10.9 times more of anthracycline compounds as compared with the initial strain. All the mutants synthesized 2.6-28.3 and 1.6-20.7 times more of DNR and DXR, respectively, than the initial strain. The results obtained prove that it is expedient to search for the strains with high production of DXR among DNR1-mutants, while DXRr-mutants are promising for choosing the producers of the both antibiotics.

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis.

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.

The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5.

The biosynthesis of daunorubicin and its precursors proceeds via the condensation of nine C-2 units derived from malonyl-CoA onto a propionyl starter moiety. The daunorubicin polyketide biosynthesis gene cluster of Streptomyces sp. strain C5 has two unique open reading frames, dpsC and dpsD, encoding, respectively, a fatty acid ketoacyl synthase (KAS) III homologue that is lacking an active-site cysteine and a proposed acyl-CoA:acyl carrier protein acyltransferase. The two genes are positioned directly downstream of dpsA and dpsB which encode the alpha and beta components of the type II KAS, respectively. Expression of the dpsABCDEFGdauGI genes in Streptomyces lividans resulted in the formation of aklanonic acid, the first stable chromophore of the daunorubicin biosynthesis pathway. Deletion of dpsC, but not dpsD, from this gene set resulted in the formation of desmethylaklanonic acid, derived from an acetyl-CoA starter unit, and aklanonic acid, derived from propionyl-CoA, in a 60:40 ratio. Thus, DpsC contributes to the selection of propionyl-CoA as the starter unit but does not alone dictate it. A dpsCD deletion mutant of Streptomyces sp. strain C5 (C5VR5) still produced daunorubicin but, more significantly, anthracycline and anthracyclinone derivatives resulting from the use of acetyl-CoA as an alternative starter moiety. Expression of dpsC, but not dpsD, in mutant C5VR5 restored the wild-type phenotype. Among the new compounds was the new biosynthesis product feudomycin D. These results suggest that in the absence of DpsC, the daunorubicin PKS complex behaves promiscuously, utilizing both acetyl-CoA (ca. 60% of the time) and propionyl-CoA (ca. 40%) as starter units. The fact that DpsC is not required for initiation with propionyl-CoA is significant, as the information must then lie in other components of the PKS complex. We propose to call DpsC the propionyl starter unit "fidelity factor."

Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis.

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, epsilon-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed by dnrE (mutants SPAK and SPMAG), dnrS (SPFS and SPRHO) and doxA (SPDHC) gene products.