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.     

Rapid purification and biochemical characterization of glucose kinase from Streptomyces peucetius var. caesius

Glucose kinase catalyzes the ATP-dependent phosphorylation of glucose. Streptomyces peucetius var. caesius glucose kinase was purified 292-fold to homogeneity. The enzyme has cytosolic localization and is composed of four identical subunits, each of 31 kDa. The purified enzyme easily dissociates into dimers. However, in the presence of 100 mM glucose the enzyme maintains its tetrameric form. Maximum activity was found at 42 degrees C and pH 7.5. Isoelectric focusing of the enzyme showed a pl of 8.4. The N- and C-terminal amino acid sequences were MGLTIGVD and VYFAREPDPIM, respectively. The kinetic mechanism of S. peucetius var. caesius glucose kinase appears to be a rapid equilibrium ordered type, i.e., ordered addition of substrates to the enzyme, where the first substrate is d-glucose. The K(m) values for d-glucose and MgATP(2-) were 1.6 +/- 0.2 and 0.8 +/- 0.1 mM, respectively. Mg(2+) in excess of 10 mM inhibits enzyme activity.     

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.     

Use of a protoplasting method for altering the qualitative and quantitative composition of the anthracycline complex in Streptomyces peucetius var. caesius

Conditions for efficient regeneration in mutant strains of the doxorubicin-producing organism Str. peucetius var. caesius were developed. The effect of the protoplast regeneration on changes in the proportion of the components of the anthracycline complex produced by these strains was shown. Variants with doxorubicin productivity 2 times higher than that of the parent strain were isolated.     

Modeling and simulation of Streptomyces peucetius var. caesius N47 cultivation and epsilon-rhodomycinone production with kinetic equations and neural networks

This study focuses on comparing different kinetic growth models and the use of neural networks in the batch cultivation of Streptomyces peucetius var. caesius producing epsilon-rhodomycinone. Contois, Monod and Teissier microbial growth models were used as well as the logistic growth modeling approach, which was found best in the simulations of growth and glucose consumption in the batch growth phase. The lag phase was included in the kinetic model with a CO2 trigger and a delay factor. Substrate consumption and product formation were included as Luedeking-Piret and logistic type equations, respectively. Biomass formation was modeled successfully with a 6-8-2 network, and the network was capable of biomass prediction with an R2-value of 0.983. Epsilon-rhodomycinone production was successfully modeled with a recursive 8-3-1 network capable of epsilon-rhodomycinone prediction with an R2-value of 0.903. The predictive power of the neural networks was superior to the kinetic models, which could not be used in predictive modeling of arbitrary batch cultivations.     

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (～66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (～44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.     

The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.     

广西细叶云南松群系的初步研究

细叶云南松是云南松原种从中亚热带温凉的云南高原向东迁移,适应南亚热带于热河谷的一个变种。它广泛分布在南盘江下游两岸海拔300-1600米的丘陵山地,常常构成大面积的天然森林,群落类型多种多样。它们的形成和分布与所在地的气候、地貌、土壤和人为生产活动特别是垦殖和烧山的影响有密切的联系。本文比较详尽地划分和论述了这个区域广西范围内细叶云南松群系各个群丛的基本特点和分布规律,指出了它们的演替趋势和经营管理应采取的主要措施。     

The differential effects produced by daunomycin and adriamycin on RNA, polynucleotides, single stranded, supercoiled DNA, and nucleosomes

Terbium, a sensitive probe whose fluorescence is strongly enhanced when bound to unpaired guanine and xanthine bases, has been employed to study the effects of adriamycin and daunomycin on a variety of nucleotide substrates. After treatment with either drug at concentrations of less than or equal to 1:500, the fluorescence of the probe was substantially abrogated. Daunomycin, however, produced a markedly greater effect than adriamycin with rRNA, linear calf thymus DNA, and polyriboguanylic acid. The difference between the drugs was experimentally significant, suggesting that changing the C9 side group from a methyl (daunomycin) to an alcohol (adriamycin) may result in a changed base sequence specificity. The distinction was also evident when changes in electrophoretic mobility of supercoiled and nucleosomal DNA was monitored, but only at much higher (1:25) drug:DNA ratios.     

Propionate precursors in the biosynthesis of daunomycin and adriamycin: A 13C nuclear magnetic resonance study

A ¹³C-NMR study of the biosynthesis of daunomycin adriamycin from propionate[1-¹³C] has been carried out in cultures of Streptomyces peucetius var. caesius. Results give direct support for the postulate that a propionate ‘starter’ is involved in the biosynthesis of both metabolites.     

Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5.

We recently described the isolation and sequence analysis of the daunomycin polyketide synthase biosynthesis genes of Streptomyces sp. strain C5 (J. Ye, M. L. Dickens, R. Plater, Y. Li, J. Lawrence, and W. R. Strohl, J. Bacteriol. 176:6270-6280, 1994). Contiguous to the daunomycin polyketide synthase biosynthesis gene region in Streptomyces sp. strain C5 are four additional genes involved in daunomycin biosynthesis, two of the products of which show similarity to different types of methyltransferases. The dauC gene, encoding aklanonic acid methyltransferase (AAMT), complements dauC-blocked mutants of Streptomyces sp. strain C5, restores in vitro AAMT activities to the mutant strains, and confers in vitro AAMT activity on Streptomyces lividans. Partial purification through gel filtration, followed by photoaffinity labeling of enriched AAMT with S-adenosyl-L-[3H-methyl]methionine, indicates that AAMT is a homodimer with an M(r) of ca. 48,000 (subunit M(r) of ca. 24,000), which corresponds with the size of the deduced gene product. The dauD gene, encoding aklanonic acid methyl ester cyclase, is divergently arranged with respect to dauC. Immediately downstream and apparently translationally coupled with dauD is the dauK gene, encoding carminomycin 4-O-methyltransferase. The dauK gene confers in vitro carminomycin 4-O-methyltransferase activity on S. lividans and is nearly identical to a similar gene isolated from Streptomyces peucetius and characterized. Directly downstream of dauK lies a gene encoding a deduced protein that is similar to the methyl esterases.     

Blidingia minima var. stolonifera var. nov. (Ulvales, Chlorophyta) from British Columbia: systematics, life history and morphogenesis

Blidingia minima var. stolonifera var. nov. is described from Vancouver, British Columbia. In previous literature this variety may have been confused with B. chadefaudii and B. minima var. minima. The new variety is part of the B. minima species complex in which spores germinate by evacuating the original spore and forming a germ tube. B. minima var. stolonifera is characterized by the development of marginal filaments on the basal disc that have one to five colourless cells and terminate in a cell from which a new disc grows. Colourless cells are devoid of chloroplasts and nuclei, and contain only small remnants of cytoplasm. The runner system is considered a mechanism of vegetative growth and propagation that enables a disc to cover a large amount of substratum before producing erect, unbranched thalli. In field and in culture, B. minima var. stolonifera typically reproduces by means of quadriflagellate zoospores; however, three plants from Vancouver formed biflagellate spores. These germinated poorly and developed into highly irregular, branched thalli.     

Photogeneration of superoxide by adriamycin and daunomycin. An electron spin resonance and spin trapping study

The hydroxyl and superoxide anion spin adducts of DMPO and 4-MePyBN, respectively, were obtained during photoirradiation of adriamycin and daunomycin solutions with visible light. Ethanol and dimethyl sulfoxide did not scavenge hydroxyl radicals in the photoirradiated drug solutions. Furthermore, the hydroxyl-DMPO spin adduct is not formed in the photolysis of air-free drug solutions, indicating that hydroxyl radicals are not directly produced in the photochemical reactions. Instead, the observed hydroxyl-DMPO is formed from the decay of the superoxide anion-DMPO spin adduct. The mechanism for generating the superoxide anion radical appears to be a direct electron transfer from the photoexcited adriamycin and daunomycin to dissolved oxygen.     

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.     

Release of iron from ferritin by cardiotoxic anthracycline antibiotics

The use of the extremely effective anthracycline antitumor drugs adriamycin and daunomycin is limited by a severe, dose-dependent cardiomyopathy. Anthracycline-induced toxicity has been proposed to involve iron-dependent oxidative damage to biological macromolecules yet little is known regarding the availability of physiologic iron. We now report that, in the presence of NADPH-cytochrome P-450 reductase, these drugs undergo redox cycling to generate superoxide which mediates a slow, reductive release of iron from ferritin, the major intracellular iron storage protein. Anaerobically, the semiquinone free radical forms of adriamycin and daunomycin catalyze a very rapid, extensive release of iron from ferritin. In contrast, diaziquone, an aziridinyl quinone antitumorigenic agent which is less cardiotoxic, is unable to release iron from ferritin. Thus, the present studies suggest that the cardiomyopathy observed with the anthracyclines, and perhaps their antineoplastic activity as well, may be related to their ability to delocalize tissue iron, thereby contributing to the formation of strong oxidants capable of damaging critical cellular constituents.     

Purification and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes.

TDP-D-glucose 4,6-dehydratase, which converts TDP-D-glucose to TDP-D-4-keto-6-deoxyglucose, was purified to near-homogeneity from the daunorubicin and baumycin-producing organism Streptomyces sp. C5 (968-fold purification with a 41% recovery), and from the daunorubicin producer Streptomyces peucetius ATCC 29050 (1000-fold purification with a 37% recovery). The TDP-D-glucose 4,6-dehydratases from Streptomyces sp. C5 and S. peucetius were determined by SDS-PAGE and HPLC gel filtration to be homodimers with subunit relative molecular masses of 39,000 and 36,000, respectively. For the enzymes from both organisms, negligible activity was observed in the absence of added NAD+, or when ADP-glucose, ADP-mannose, GDP-mannose, UDP-glucose or UDP-galactose was substituted for TDP-D-glucose as substrate. For the enzyme from Streptomyces sp. C5, the K'm values for NAD+ and TDP-D-glucose were 19.2 microM and 31.3 microM, respectively. The V'max for TDP-D-glucose was 309 nmol min-1 (mg protein)-1. For the S. peucetius enzyme, the K'm values for NAD+ and TDP-D-glucose were 20.1 microM and 34.7 microM, respectively. V'max values were 180 nmol min-1 (mg protein)-1 for NAD+ and 201 nmol min-1 (mg protein)-1 for TDP-D-glucose. TDP was a good inhibitor of TDP-D-glucose 4,6-dehydratase from both organisms. The N-terminal amino acid sequence of the TDP-D-glucose 4,6-dehydratase from S. peucetius and from the erythromycin producer, Saccharopolyspora erythraea, were similar, whereas the enzyme from Streptomyces sp. C5 contained a different N-terminal amino acid sequence from either of the other two enzymes.     

Transformation and transfection of anthracycline-producing streptomycetes.

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

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.     

Adriamycin and daunomycin induce interstrand DNA crosslinks in HeLa S3 cells

By using a new mild procedure for detecting DNA crosslinks it has been shown that adriamycin and daunomycin are able to form interstrand DNA crosslinks in HeLa cells. This effect seems to be preceded by transformation of the parent antibiotics in the cell to active forms. In addition, interstrand DNA crosslinks formed by adriamycin and daunomycin were found to be temperature- and alkali-labile.