Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene.

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that contains dpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and the doxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacing dnrU, dnrV, doxA, or dpsH with mutant alleles and of doxA overexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379-2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316-7321, 1996) triple mutant. Overexpression of doxA in a doxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxA jointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.

Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine.

Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis.

Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis.

The dnrQS genes from the daunorubicin producer Streptomyces peucetius were characterized by DNA sequencing, complementation analysis, and gene disruption. The dnrQ gene is required for daunosamine biosynthesis, and dnrS appears to encode a glycosyltransferase for the addition of the 2,3,6-trideoxy-3-aminohexose, daunosamine, to epsilon-rhodomycinone.

Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus.

Sequence analysis of the dnrR2 locus from the cluster of daunorubicin biosynthesis genes in Streptomyces peucetius ATCC 29050 has revealed the presence of two divergently transcribed open reading frames, dnrN and dnrO. The dnrN gene appears to encode a response regulator protein on the basis of conservation of the deduced amino acid sequence relative to those of known response regulators and the properties of the dnrN::aphII mutant. Surprisingly, amino acid substitutions (glutamate and asparagine) at the putative site of phosphorylation (aspartate 55) resulted in a reduction rather than a complete loss of DnrN activity. The deduced DnrO protein was found to be similar to the Streptomyces glaucescens tetracenomycin C resistance gene repressor (TcmR) and to two Escherichia coli repressors, the biotin operon repressor (BirA) and the tetracycline resistance gene repressor (TetR). The dnrN::aphII mutation was suppressed by introduction of the dnrI gene on a plasmid. Since the introduction of dnrN failed to restore antibiotic production to a dnrI::aphII mutant, these data suggest the presence of a regulatory cascade in which dnrN activates the transcription of dnrI, which in turn activates transcription of the daunorubicin biosynthesis genes.

Genetic control of polyketide biosynthesis in the genus Streptomyces.

The genetic control of polyketide metabolite biosynthesis in Streptomyces sp. producing actinorhodin, daunorubicin, erythromycin, spiramycin, tetracenomycin and tylosin is reviewed. Several examples of positively-acting transcriptional regulators of polyketide metabolism are known, including some two-component sensor kinase-response regulator systems. Translational and posttranslational control mechanisms are only briefly mentioned since very little is known about either of these processes. Examples of how enzyme levels and substrate supply affect polyketide metabolism also are discussed.

Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius.

Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR1 segment caused a 10-fold overproduction of epsilon-rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9-kb dnrR2 segment increased production of both epsilon-rhodomycinone and daunorubicin 10- and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin-sensitive mutant of S. peucetius subsp. caesius ATCC 27952. Analysis of the sequence of the dnrR1 fragment revealed the presence of two closely situated open reading frames, dnrI and dnrJ, whose deduced products exhibit high similarity to the products of several other Streptomyces genes that have been implicated in the regulation of secondary metabolism. Insertional inactivation of dnrI in the ATCC 29050 strain with the Tn5 kanamycin resistance gene abolished epsilon-rhodomycinone and daunorubicin production and markedly decreased resistance to daunorubicin. Sequence comparison between the products of dnrIJ and the products of the Streptomyces coelicolor actII-orf4, afsR, and redD-orf1 genes and of the Streptomyces griseus strS, the Saccharopolyspora erythraea eryC1, and the Bacillus stearothermophilus degT genes reveals two families of putative regulatory genes. The members of the DegT, DnrJ, EryC1, and StrS family exhibit some of the features characteristic of the protein kinase (sensor) component of two-component regulatory systems from other bacteria (even though none of the sequences of these four proteins show a significant overall or regional similarity to such protein kinases) and have a consensus helix-turn-helix motif typical of DNA binding proteins. A helix-turn-helix motif is also present in two of the proteins of the other family, AfsR and RedD-Orf1. Both sets of Streptomyces proteins are likely to be trans-acting factors involved in regulating secondary metabolism.

Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius.

Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomyces peucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actI/tcmIa and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a cosmid vector established that the DNA represented three nonoverlapping regions of the S. peucetius subsp. caesius genome. These three regions plus an additional one that hybridized to the same probes are present in the S. peucetius genome, as reported previously (K. J. Stutzman-Engwall and C. R. Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135-3139, 1989). Functional analysis of representative clones from some of these regions in S. lividans, S. peucetius ATCC 29050, S. peucetius subsp. caesius ATCC 27952, and two of its blocked mutants (strains H6101 and H6125) showed that many of the antibiotic production genes reside in the region of DNA represented by the group IV clones. This conclusion is based on the production of epsilon-rhodomycinone, a key intermediate of the daunorubicin pathway, in certain S. lividans transformants and on the apparent complementation of mutations that block daunorubicin biosynthesis in strains H6101 and H6125. Some of the transformants of strains 29050, 27952, and H6125 exhibited substantial overproduction of epsilon-rhodomycinone and daunorubicin.

Frequent loss and restoration of antibiotic production by Streptomyces lasaliensis.

Antibiotic nonproducing variants of Streptomyces lasaliensis NRRL 3382R, which makes the polyether antibiotic lasalocid A (Las) and the quinoxaline antibiotic echinomycin (Ech), arose at a frequency of 3-11% after treatment with three different mutagens or regeneration of protoplasts compared with a spontaneous frequency of less than 0.1%. Cosynthesis of lasalocid A was not observed upon testing a large number of Las- mutants in different pair-wise combinations, nor did these mutants accumulate probable intermediates of lasalocid A biosynthesis. These results suggest that loss of the las genes or their expression is induced at a high frequency by mutagenic treatments. In fusions of protoplasts of a strain with the las+ ech+ spo+ nic-1 rif-3 markers with strains bearing the Las- LasS Ech- Bld- (or spo+) str-1 markers, Las+ Ech+ Spo+ StrR progeny were produced at a 61-89% frequency compared with a 1-9% frequency of StrR antibiotic producing progeny with the nic-1 or rif-3 genotypes. The more frequent restoration of antibiotic production than prototrophy or rifampicin sensitivity indicates that these antibiotic characters did not behave as normal chromosomal markers. Therefore the genetic instability might be due to the involvement of a plasmid in antibiotic production. The apparent lack of infectious transfer of the Las+ character to Las- parents in conjugal matings between the few strains tested and no correlation between the presence of a large plasmid, pKSL, and lasalocid A production in several strains of S. lasaliensis do not favor the latter hypothesis, but they do not conclusively disprove it. Consequently, we suggest that a plasmid or another mobile genetic element is controlling antibiotic production in S. lasaliensis.

Oxidative ring fission of the naphthoquinones lapachol and dichloroallyl lawsone by Penicillium notatum.

The naphthoquinones lapachol and dichloroallyl lawsone readily undergo oxidative ring fission when incubated with several fungi and streptomycetes. Penicillium notatum was employed to produce the ring fission product of dichloroallyl lawsone which was isolated and characterized by spectral analyses and chemical synthesis. The mechanism of oxidative ring fission of lapachol was studied by growing P. notatum cultures in an 18O2 atmosphere. Mass spectral analysis of the isolated and labeled metabolite indicates that ring fission occurs via a monooxygenase pathway most probably involving an epoxide intermediate.