Cloning of the genes encoding thymidine diphosphoglucose 4,6-dehydratase and thymidine diphospho-4-keto-6-deoxyglucose 3,5-epimerase from the erythromycin-producing Saccharopolyspora erythraea

Kj, Linton; Bw, Jarvis; Cr, Hutchinson

doi:10.1016/0378-1119(94)00809-7

Cited by 47 publications

(38 citation statements)

References 24 publications

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“…Among bacteria, these chromosomes are only smaller S . erythraea physical-genetic map Donadio et al (1996) Donadio et al (1996) O'Neill et al (1995) Donadio et al (1993) Linton et al (1995 S. Zotslav (unpublished) This study Brown et al (1990) Linton et at. (1995 " Includes the entire -56 kb erythromycin-biosynthetic gene cluster.…”

Section: Discussionmentioning

confidence: 80%

“…t g d h and kde are adjacent to one another on the chromosome (Linton et al, 1995). *pSE101 and pSE211 integrate into the S. erythraea NRRL 2338 chromosome at two different attB sites on AseI-B and DraI-I1 and VI.…”

Section: Discussionmentioning

confidence: 99%

“…Functions for the majority of the predicted genes have been proposed (Donadio et al, 1993;Summers et al, 1997). Several additional genes thought to be involved in erythromycin biosynthesis have been identified and characterized (Hsieh & Kolattukudy , 1994 ;Linton et With the recent development of high-resolution PFGE, physical mapping of large ( > 5 Mb) bacterial chromosomes has become feasible. Recently, the physical maps of the -8 Mb chromosomes of several Streptomyces spp., which are closely related to Saccharopolyspora, have been reported (Kieser et al, 1992;LeBlond et al, 1993LeBlond et al, , 1996Lezhava et al, 1995;Pandza et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

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Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea

1998

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A physical map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes Asel and Dral. The map was constructed by a variety of methods including linking clone analysis, cross-hybridizations using labelled macrorestriction fragments, gene probing, two-dimensional PFGE and restriction enzyme site generation. Analysis of the individual macrorestriction patterns of the 17 Asel-, 6 Drab and 22 AsellDral-digested fragments indicated a chromosome size of about 8 Mb. Linking clones for five contiguous Asel fragments were obtained, covering 32% of the chromosome. The linkage of an additional eight Asel fragments was aided by the finding that the rRNA operons of S. erythraea contain an Asel site within the 165 ( r a ) gene. Generation of 5. erythraea strains that contain additional DraI sites within selected Asel fragments, followed by PFGE analysis and Southern hybridization to determine specific linkages, facilitated the completion of the Asel map. The entire DraI map was constructed by gene probing and cross-hybridizations. PFGE analysis of agarose-embedded DNA prepared in either the presence or absence of proteinase K suggested that the S. erythraea NRRL 2338 chromosome is linear. A total of 15 genes or gene clusters were mapped to specific Asel and Dral fragments, including the erythromycin-biosynthetic gene cluster and the rRNA operons.

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Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea

1998

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“…DNR biosynthesis is unique in this regard since a nonfunctional gene is present, but another gene product from outside the biosynthetic gene cluster complements the missing activity. In erythromycin biosynthesis, the cluster of production genes, as currently defined, lacks genes encoding glucose-1 -phosphate thymidylyl transferase and TDP-D-glucose 4,6-dehydratase, but genes for these enzymes have been found elsewhere in S. eytbraea (Linton et al, 1995 The unglycosylated intermediate RHO is approximately 10-fold more prevalent than DNR when S. pegcetizls is grown in GPS medium (Dekleva et al, 1985). Therefore, it appears quite likely that one of the steps of daunosamine biosynthesis or attachment is rate-limiting.…”

Section: Discussionmentioning

confidence: 99%

The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-production gene cluster

1996

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A 2.7 kb BamHl fragment of the daunorubicin biosynthetic cluster in Streptomyces peucetius ATCC 29050 was shown to contain two ORFs, dnrL and dnfM# whose deduced products exhibit a high sequence similarity to a number of glucose-l-phosphate thymidylyl transferases and TDP-D-glucose dehydratases, respectively. Although these genes were believed to be necessary for the synthesis of the deoxyaminosugar, daunosamine, a constituent of daunorubicin, the dnrM gene contains a frameshift in the DNA sequence that causes the premature termination of translation. A gene encoding another TDP-glucose 4#6=dehydratase, previously isolated from 5. peucetius, was identified by PCR amplification of genomic DNA. The presence of this gene explains why a dnrM::aphll mutation did not block daunorubicin production.

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“…A25110), and DnrL (68.3% identity and 79.7% similarity) from the daunorubicin pathway (6). The MtmE protein strongly resembled TDP-D-glucose 4,6-dehydratases from different antibiotic pathways: StrE (69.2% identity and 81.4% similarity) from the streptomycin pathway (13); TylA2 (62.8% identity and 73.8% similarity) from the tylosin pathway (10); GraE (60% identity and 75% similarity) from the granaticin pathway (1); and Gdh (65.6% identity and 76.2% similarity), a dehydratase from the erythromycin producer Saccharopolyspora erythraea, which has been shown not to be involved in antibiotic biosynthesis (7). The MtmE protein shows an amino acid sequence (GGAGFIG) close to the amino terminus corresponding to the motif GXGXXG (X representing any amino acid), which has been described as a ␤␣␤ fold with an NAD binding motif (17).…”

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confidence: 99%

Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin

et al. 1997

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Two genes (mtmD and mtmE) were cloned and sequenced from the mithramycin producer Streptomyces argillaceus. Comparison with proteins in databases and enzymatic assays after expression in Escherichia coli showed that they encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. The mtmD gene was inactivated by gene replacement, generating a nonproducing mutant that accumulates a tetracyclic compound designated premithramycinone. The identification of premithramycinone reveals new aspects of the mithramycin biosynthetic pathway and suggests that at least some glycosylations occur before breakage of the fourth ring.Mithramycin (also designated aureolic acid, plicamycin, mithracin, LA7017, and A2371) is an antitumor drug synthesized by different actinomycete species and has clinical application in the treatment of several tumors (14). It also possesses antibiotic activity against gram-positive but not gram-negative bacteria. Structurally, it is an aromatic polyketide containing a three-ring chromophoric aglycon derived from the condensation of 10 acetates (11). Genes for a type II polyketide synthase were cloned and sequenced from a mithramycin producer, Streptomyces argillaceus; their involvement in mithramycin biosynthesis has been demonstrated by insertional inactivation (9), and evidence in favor of the biosynthesis of the aglycon through a single polyketide backbone instead of two has been provided elsewhere (3). The mithramycin aglycon (mithramycinone) is glycosylated at positions 6 and 2 with disaccharide and trisaccharide moieties, respectively. These sugars belong to the large family of the 6-deoxyhexoses (6DOHs), which are synthesized through biosynthetic pathways that have early biosynthetic steps in common (8, 12). In 6DOH biosynthesis, first glucose-1-phosphate is converted into TDP-D-glucose by the action of a glucose-1-phosphate:TTP thymidylyl transferase. Then, a TDP-D-glucose 4,6-dehydratase is responsible for the conversion of TDP-D-glucose into TDP-D-4-keto-6-deoxyglucose. Further action of a 3,5-epimerase can direct TDP-D-4-keto-6-deoxyglucose into the L series of 6DOHs; in contrast, if there is no participation of an epimerase, the final sugars will be D-6DOHs. Further modifications in both L-and D-6DOHs via isomerization, methylation, reduction, dehydration, etc., can produce a great variety of 6DOHs, which are part of the structures of different actinomycete metabolites (12, 18).Here we report the cloning, sequencing, and heterologous expression of two genes involved in the biosynthesis of the sugar moieties of the antitumor drug mithramycin. These genes (mtmD and mtmE) encode a glucose-1-phosphate:TTP thymidylyl transferase and a TDP-D-glucose 4,6-dehydratase, respectively. We also report the construction of a non-mithra-FIG. 1. Schematic representation of the region sequenced from cosAR7 and the different deduced ORFs and its location with respect to the mithramycin polyketide synthase (PKS). The bar indicates the region showing homology to t...

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Cloning of the genes encoding thymidine diphosphoglucose 4,6-dehydratase and thymidine diphospho-4-keto-6-deoxyglucose 3,5-epimerase from the erythromycin-producing Saccharopolyspora erythraea

Cited by 47 publications

References 24 publications

Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea

Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea

The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshift mutation that is suppressed by another locus outside of the daunorubicin-production gene cluster

Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin

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