Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn–Arg–hOrn–hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.     

Precursor for biosynthesis of sugar moiety of doxorubicin depends on rhamnose biosynthetic pathway in Streptomyces peucetius ATCC 27952

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 contains a TDP-D-glucose 4,6-dehydratase gene, dnmM, that is putatively involved in the biosynthesis of daunosamine, but the gene contains a frameshift in the DNA sequence that would cause premature termination of translation. In pursuit of another TDP-D-glucose 4,6-dehydratase in S. peucetius, a homologue gene, rmbB, was found, whose deduced product exhibits high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was located within a putative rhamnose biosynthetic gene cluster at another locus in the genome. RmbB was verified to be a functional TDP-D-glucose 4,6-dehydratase by enzyme assay as it catalyzed the conversion of TDP-D-glucose into TDP-4-keto-6-deoxy-D-glucose. Inactivation of rmbB in the S. peucetius genome abolished the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restored the doxorubicin production. Hence, rmbB provides TDP-4-keto-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin.     

Expression profiling of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.     

Exploration of two epimerase homologs in Streptomyces peucetius ATCC 27952

Streptomyces peucetius ATCC 27952 is a potent producer of the therapeutically important antitumor drug, doxorubicin. S. peucetius contains two deoxythymidine diphospho (dTDP)-4-keto-6-deoxyglucose 3,5-epimerase-encoding genes, dnmU and rmbC, in its genome. While dnmU from the doxorubicin biosynthesis gene cluster is involved in the biosynthesis of dTDP-l-daunosamine, rmbC is involved in the biosynthesis of dTDP-l-rhamnose, a precursor of cell wall biosynthesis. The proteins encoded by dnmU and rmbC share 47 % identity and 64 % similarity with each other. Both enzymes converted the same substrate, dTDP-4-keto-6-deoxy-d-glucose, into dTDP-4-keto-l-rhamnose in vitro. However, when disruption of dnmU or rmbC was carried out, neither gene in S. peucetius compensated for each other’s loss of function in vivo. These results demonstrated that although dnmU and rmbC encode for similar functional proteins, their native roles in their respective biosynthetic pathways in vivo are specific and independent of one other. Moreover, the disruption of rmbC resulted in fragmented mycelia that quickly converted into gray pigmented spores. Additionally, the production of doxorubicin, a major product of S. peucetius, appeared to be abolished after the disruption of rmbC, demonstrating its pleiotropic effect. This adverse effect might have switched on the genes encoding for spore formation, arresting the expression of many genes and, thereby, preventing the production of other metabolites.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

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.     

Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius

Streptomyces peucetius var. caesius, obtained from S. peucetius, the daunomycin producing microorganism, by mutagenic treatment, differs from the parent culture by the color of the vegetative and aerial mycelia and by its antibiotic, producing ability. S. peucetius var. caesius accumulates adriamycin in submerged and aerated culture on a medium containing glucose, brewer's yeast, and inorganic, salts both in shake flasks and in stirred fementers. Isolation of the product is performed by solvent extraction, chromatography on buffered cellulose columns, and crystallization as the hydrochloride. The new antitumor agent, adriamycin, is the 14-hydroxv derivative of daunomyein.     

Exploration of geosmin synthase from <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster

Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 ± 0.4-fold enhanced production of geosmin was observed.     

Glucose kinases from Streptomyces peucetius var. caesius

Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX₍₄₎SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.     

Effect of glucose on the antibiotic activity and antibiotic resistance of Streptomyces peucetius subsp. caesius ATCC 27952-2 and its mutants]

The growth of anthracycline producer Streptomices peucetius subsp. caesius ATCC 27952-2 was inhibited by presence of glucose on complete media, containing alternative carbon sources. Amount of clones not producing antibiotic increased to 80.2 per cent along with elevation of glucose concentration in corn meal medium from 0.1 to 1.0 per cent. Mutants of S. peucetius subsp. caesius ATCC 27952-2 able to grow on complete media with 2 per cent of glucose (glr-mutants) were obtained. Glr-mutants had decreased antibiotic production in comparison with 27952-2 strain. 17 per cent of studied glr-mutants synthesized 1.6-3.1-fold quantities of anthracyclines in comparison with parental strain. Glr-mutants synthesized more biomass, although more slowly utilized glucose than strain 27952-2.     

Proteomic approach to enhance doxorubicin production in <Emphasis Type="Italic">panK</Emphasis>-integrated <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> 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.     

Isolation and characterization of stable mutants ofStreptomyces peucetius defective in daunorubicin biosynthesis

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced byStreptomyces peucetius. In this study we report isolation of stable mutants ofS. 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, ε-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed bydnrE (mutants SPAK and SPMAG),dnrS (SPFS and SPRHO) anddoxA (SPDHC) gene products.     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

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%.     

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.