Construction of a genetic map of chromosomal auxotrophic markers in Streptomyces peucetius var. caesius.

Ho, C. C.; Chye, Mee‐Len

doi:10.2323/jgam.31.231

Cited by 7 publications

(6 citation statements)

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“…Functional Analysis of Representative Clones from Each Group. To determine whether the different regions of DNA from S. peucetius have a function in the daunorubicin biosynthetic pathway, representative clones from groups I-IV were transformed into S. lividans and also into the S. peucetius H6101 and H6125 blocked mutants (16). Two approaches were taken to confirm the presence of plasmid DNA in transformed Streptomyces: plasmid DNA was isolated from the transformant and used to retransform S. lividans, or total DNA was isolated from the transformant, digested with the appropriate restriction endonuclease, transferred to a nylon membrane, and hybridized with a 32p_ labeled fragment from the plasmid.…”

Section: Resultsmentioning

confidence: 99%

“…Streptomyces lividans TK24 (13) and 1326 (14) were obtained from David Hopwood (John Innes Institute, Norwich, U.K.) and S. lividans JT46 (15) was obtained from Carton Chen (Institute of Microbiology and Immunology, National Yang Ming Medical College, Taipei, Taiwan, Republic of China). S. peucetius strain H6101, which appears to be blocked early in the daunorubicin biosynthetic pathway (16), and strain H6125, which is blocked later and accumulates E-rhodomycinone (16) Streptomyces strains were maintained on R2YE agar (18) and grown in R2YE liquid prior to chromosomal or plasmid DNA isolations and for preparing seed inoculum for fermentations. Streptomyces protoplasts were regenerated on R2YE agar.…”

Section: Methodsmentioning

confidence: 99%

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Multigene families for anthracycline antibiotic production in Streptomyces peucetius.

Stutzman-Engwall

Hutchinson

1989

Proc. Natl. Acad. Sci. U.S.A.

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Hybridization of polyketide synthase genes from heterologous Streptomyces sp. led to the identification of four unlinked regions of DNA from Streptomycespeucetus that contain genes that encode the production of the same or dosely related metabolites, some of which are intermediates of the daunorubicin pathway. DNA fragments from each region that hybridized with the heterologous polyketide synthase genes were hybridized with each other, but very little sequence similarity was observed even though at least two of the regions have similar (if not identical) functions in metabolite production. Some regions, however, do have sequence similarity with other anthracycline-producing Streptomyces sp.The anthracycline antibiotics daunorubicin and doxorubicin are important antitumor drugs used in cancer chemotherapy even though they are cardiotoxic (1) and their production cost is high because ofthe complex mixture ofmetabolites that are produced during fermentation (2). Most of the research effort in investigating these problems has focused on traditional methods of large-scale screening, mutant construction, directed biosynthesis, and analog synthesis. While progress has been made, there has been little success in reducing toxicity or determining a definitive biosynthetic pathway. A solution to these problems may be found through studies in which molecular approaches are used to investigate the organization and regulation of the biosynthetic genes for the daunorubicin/doxorubicin antibiotic pathway. Developments in gene cloning technologies for Streptomyces have made it possible to clone antibiotic biosynthetic pathway genes from one organism and to transfer the entire region, or portions of it, into other suitable hosts (3, 4). In this manner, biosynthetic genes are being characterized, antibiotic resistance genes identified, and new "hybrid" antibiotics created (3-11). With the recent report on the existence of extensive homology between putative "polyketide synthase" genes in Streptomyces sp. (12), we developed a cloning strategy to identify such genes, which encode the earliest pathway enzymes of antibiotic production, in Streptomyces peucetius by DNA hybridization using Escherichia coli-Streptomyces shuttle cosmids as cloning vectors. We describe here the isolation, identification, and characteristics of four distinct nonoverlapping regions of DNA from S. peucetius that are involved in anthracycline metabolite production. At least one region contains a daunorubicin-resistance gene. Streptomyces strains were maintained on R2YE agar (18) and grown in R2YE liquid prior to chromosomal or plasmid DNA isolations and for preparing seed inoculum for fermentations. Streptomyces protoplasts were regenerated on R2YE agar. E. coli strains were maintained and grown in LB medium (19). For S. lividans strains transformed with the vectors pNJ1 or pKC505, thiostrepton (Thio) or apramycin (Am) were added at 50 or 100 Ag/ml, respectively. S. peucetius transformants were selected and maintained with Thio (25 ug/ml) or Am (25-50 ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Multigene families for anthracycline antibiotic production in Streptomyces peucetius.

Stutzman-Engwall

Hutchinson

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…Strains H6101 and H6125 are blocked mutants of S. peucetius subsp. caesius prepared by Ho et al (10,11). According to those workers, H6125 produces no daunorubicin but accumulates e-rhodomycinone.…”

Section: Methodsmentioning

confidence: 99%

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

1990

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Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomycespeucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actIltemla and actII polyketide synthase gene probes. Restriction Daunorubicin (daunomycin) and doxorubicin (adriamycin) are commercially important antibiotics with potent antitumor activity. Daunorubicin, first isolated in 1963 from Streptomyces peucetius (6, 7), was subsequently found in a number of other Streptomyces spp. (27). Doxorubicin was isolated in 1969 from S. peucetius subsp. caesius, a mutant of the wild-type strain (2), and has important clinical applications in cancer chemotherapy (1), even though both doxorubicin and daunorubicin cause cardiotoxic side effects that are dose limiting and irreversible. The production costs of these antibiotics are high because of low titers and formation of a complex mixture of products by the producing bacteria. Therefore, a genetic study of the biosynthesis of daunorubicin and doxorubicin was undertaken in our laboratory to elucidate the organization and regulation of the biosynthetic genes, with the hope that it may also lead to overproducing strains or strains with a simpler spectrum of secondary metabolites. This work has been facilitated by the recognition that the early genes involved in polyketide biosynthesis by other streptomycetes, such as the S. coelicolor actI and actIlI genes (9, 17) and the S. glaucescens tcmIa genes (20), hybridize to the analogous genes in other polyketide producers (16) and by the fact that antibiotic genes have been found to be clustered in all of the examples studied (14).A previous report (24) from this laboratory demonstrated that the daunorubicin producer S. peucetius (ATCC 29050) contains five nonoverlapping regions of DNA that hybridized to the actIl/tcmIa or actIII probe. The properties of S. peucetius and S. lividans strains transformed with clones from four of these regions supported the belief that many of the daunorubicin production (dnr) genes resided in region IV but also raised the possibility that bona fide dnr genes or genes that influence daunorubicin production and self-resistance were present in the three other regions (24). The latter idea is best tested by examining the effects of deletions in * Corresponding author.each of these four regions on daunorubicin production and resistance.Since our preliminary evidence (24) indicated that some portion of region II had been deleted from S. peucetius subsp. caesius (ATCC 27952), we made a detailed study of the properties of cosmid clones from three nonoverlapping regions of DNA in this strain. The effects of these clones and additional clones from the wild-type 29050 strain in homologous and heterologous hosts confirm that the genes required for formation of e-rhodomycinone, a key intermediate of daunorubicin biosynthesis, are present in region IV. In addition, we demonstrated that this region contains two daunorubi...

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“…S. peucetius subsp. caesius H6101 and H6125 (21,22) were obtained from Ho Coy-Choke (University of Malaya, Kuala Lumpur, Malaysia). Escherichia coli DH5␣ and JM105 (38) were used for subcloning and single-stranded DNA preparation.…”

Section: Methodsmentioning

confidence: 99%

Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus

1995

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Sequence analysis of the dnrR 2 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.Members of the genus Streptomyces are filamentous soil bacteria which produce a wide array of secondary natural products, many of which are useful therapeutic agents (11). These bacteria also possess a complex life cycle that requires temporal differentation of morphology and physiology (9, 24). Little is known about the regulation of the processes involved in Streptomyces differentation, but it is clear that a better understanding of the regulation of antibiotic production genes at the molecular level would provide insight into the fundamental issue of temporal regulation of differentation and secondary metabolism in Streptomyces spp. This knowledge in turn should facilitate efforts to engineer strains that overproduce valuable microbial metabolites.We have been engaged in a study of the molecular biology of daunorubicin (DNR) biosynthesis, resistance, and regulation in Streptomyces peucetius ATCC 29050. DNR and doxorubicin (14-hydroxy-DNR) (Fig. 1) are commercially important chemotherapeutic agents, and their study has proved to be a useful vehicle with which to address fundamental issues of Streptomyces molecular biology.Previous reports from this laboratory (35, 43) have demonstrated that the DNR producer S. peucetius possesses two DNA segments of approximately 2 kb, dnrR 1 and dnrR 2 , both of which restore DNR production to a putative regulatory mutant (S. peucetius H6101) even though the two segments are separated from each other by approximately 12.4 kb (43). These two DNA fragments also caused overproduction of DNR and ε-rhodomycinone (RHO), an intermediate of DNR biosynthesis (Fig. 1), when introduced into wild-type and mutant strains (43). In addition, only the dnrR 2 segment conferred high-level DNR resistance to the H6101 self-sensitive mutant (43). These results sug...

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Construction of a genetic map of chromosomal auxotrophic markers in Streptomyces peucetius var. caesius.

Cited by 7 publications

References 2 publications

Multigene families for anthracycline antibiotic production in Streptomyces peucetius.

Multigene families for anthracycline antibiotic production in Streptomyces peucetius.

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

Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus

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