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1.
J Trace Elem Med Biol ; 32: 195-9, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26302929

ABSTRACT

The German, Austrian and Swiss nutrition societies are the joint editors of the 'reference values for nutrient intake'. They have revised the reference values for the intake of selenium and published them in February 2015. The saturation of selenoprotein P (SePP) in plasma is used as a criterion for the derivation of reference values for selenium intake in adults. For persons from selenium-deficient regions (China) SePP saturation was achieved with a daily intake of 49µg of selenium. When using the reference body weights the D-A-CH reference values are based upon, the resulting estimated value for selenium intake is 70µg/day for men and 60µg/day for women. The estimated value for selenium intake for children and adolescents is extrapolated using the estimated value for adults in relation to body weight. For infants aged 0 to under 4 months the estimated value of 10µg/day was derived from the basis of selenium intake via breast milk. For infants aged 4 to under 12 months this estimated value was used and taking into account the differences regarding body weight an estimated value of 15µg/day was derived. For lactating women compared to non-lactating women a higher reference value of 75µg/day is indicated due to the release of selenium with breast milk. The additional selenium requirement for pregnant women is negligible, so that no increased reference value is indicated.


Subject(s)
Feeding Behavior , Selenium/pharmacology , Age Distribution , Humans , Reference Values
2.
Dtsch Med Wochenschr ; 137(8): 389-93, 2012 Feb.
Article in German | MEDLINE | ID: mdl-22334365

ABSTRACT

The relative contribution of nutrition-related chronic diseases to the total disease burden of the society and the healthcare costs have risen continuously over the last decades. This challenge requires to explore and use the potential of dietary prevention of diseases such as obesity, type 2 diabetes, dyslipidaemia, cardiovascular disease and cancer. This evidence-based guideline systematically assessed the potential role of carbohydrates in the primary prevention of these diseases. The major findings were: a high consumption of sugar-sweetened beverages increases the risk of obesity and type 2 diabetes (strength of evidence: probable), whereas a high dietary fibre intake, mainly from whole-grain products, reduces the risk of obesity, type 2 diabetes, dyslipidaemia, hypertension, coronary heart disease and colorectal cancer (strength of evidence: probable and convincing, respectively). The practical consequences for current dietary recommendations are presented.


Subject(s)
Diet, Carbohydrate-Restricted/statistics & numerical data , Dietary Carbohydrates/therapeutic use , Nutrition Disorders/epidemiology , Nutrition Disorders/prevention & control , Practice Guidelines as Topic , Germany/epidemiology , Humans , Nutrition Disorders/diet therapy , Risk Assessment , Risk Factors
3.
Genetika ; 46(5): 604-9, 2010 May.
Article in English | MEDLINE | ID: mdl-20583594

ABSTRACT

LanK is TetR-like regulatory protein recently shown to regulate the export and glycosylation of landomycins in Streptomyces cyanogenus S136. Here, several properties of the lanK-mediated regulation were deciphered. LanK seems to function as oligomer as evident from experiments in vitro. In vivo, it is able to recognize various landomycins with altered aglycon structure and the minimal concentration of landomycin A sensed by LanK lies in low nanomolar range. Coexpression studies showed that the positive regulatory gene lanI upregulates lanK-dependent lan genes once the negative LanK-regulation is cancelled. Gene lanK can be useful for the construction of biosensor strains for sensitive and specific identification of producers of landomycin-like molecules with long glycosidic chains.


Subject(s)
Aminoglycosides/biosynthesis , Bacterial Proteins/metabolism , Streptomyces/metabolism , Aminoglycosides/genetics , Bacterial Proteins/genetics , Biological Transport/physiology , Biosensing Techniques , Protein Multimerization , Streptomyces/genetics
4.
J Biotechnol ; 130(1): 32-8, 2007 May 31.
Article in English | MEDLINE | ID: mdl-17434221

ABSTRACT

A strain (S. fradiae Delta urdQ/R) with mutations in urdQ and urdR encoding a dTDP-hexose-3,4-dehydratase and a dTDP-hexose-4-ketoreductase, respectively, produces a new urdamycin analogue (urdamycin X) with changes in the polyketide structure. The structure of urdamycin X has been elucidated by NMR spectroscopy. Urdamycin X was not detectable, even in small amounts, in either S. fradiae Delta urdQ, in S. fradiae DeltaurdR or in S. fradiae A0, a mutant lacking all glycosyltransferase genes. Complementation of S. fradiae Delta urdQ/R restored urdamycin A production indicating that the mutations did not cause any polar effect.


Subject(s)
Biotechnology , Streptomyces/genetics , Streptomyces/metabolism , Aminoglycosides/chemistry , Aminoglycosides/metabolism , Anthraquinones/chemistry , Anthraquinones/metabolism , Gene Expression Regulation, Bacterial , Glycosylation , Macrolides/chemistry , Macrolides/metabolism , Mutation , Plasmids , Streptomyces/classification
5.
Genetika ; 42(5): 595-601, 2006 May.
Article in English | MEDLINE | ID: mdl-16808239

ABSTRACT

Mobilizable shuttle plasmids containing the origin of transfer (oriT) region of plasmid F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPalpha) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Streptomyces. The conjugative system of the IncPalpha plasmids was shown to be most effective in conjugative transfer, giving peak values of (2.7 +/- 0.2) x 10(-2) S. lividans TK24 exconjugants per recipient cell. To assess whether the mating-pair formation system or the DNA-processing apparatus of the IncPalpha plasmids is crucial in conjugative transfer, an assay with an IncQ-based mobilizable plasmid (RSF1010) specifying its own DNA-processing system was developed. Only the IncPalpha plasmid mobilized the construct to S. lividans indicating that the mating-pair formation system is primarly responsible for the promiscuous transfer of the plasmids between E. coli and Streptomyces. Dynamic of conjugative transfer from E. coli to S. lividans was investigated and exconjugants starting from the first hour of mating were obtained.


Subject(s)
Conjugation, Genetic , Escherichia coli/genetics , Streptomyces/genetics , Anti-Bacterial Agents/biosynthesis , F Factor/genetics , Streptomyces/metabolism , Streptomyces lividans/genetics , Streptomyces lividans/metabolism
6.
Antimicrob Agents Chemother ; 50(6): 2113-21, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16723573

ABSTRACT

The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic alpha-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that alpha-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Genes, Bacterial , Gram-Positive Bacteria/drug effects , Multigene Family/genetics , Polyenes/metabolism , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Base Sequence , Chromosomes, Bacterial , Cloning, Molecular , Culture Media/analysis , Gene Deletion , Gram-Positive Bacteria/genetics , Microbial Sensitivity Tests , Molecular Structure , Mutagenesis, Insertional , Mutation , Open Reading Frames , Plasmids , Polyenes/analysis , Polyenes/isolation & purification , Protein Structure, Tertiary , Sequence Analysis, DNA , Streptomyces aureofaciens/chemistry
7.
Genetika ; 41(5): 595-600, 2005 May.
Article in Russian | MEDLINE | ID: mdl-15977809

ABSTRACT

PCR screening of type I polyketidesynthase genes (PKS) was conducted in genomes of actinomycetes, producers of antibiotics. Some DNA fragments from the Streptomyces globisporus 1912 strain, a producer of a novel angucycline antibiotic landomycin E, were amplified. These fragments shared appreciable homology with type I PKS controlling the biosynthesis of polyene antibiotics (pymaricin and nistatin). The cloned regions were used to inactivate putative type I PKS genes in S. globisporus 1912. Strains with inactivated genes of PKS module do not differ from the original strain in the spectrum of synthesized polyketides. Apparently, these are silent genes, which require specific induction for their expression. The method of PCR screening can be used in a large-scale search for producers of new antibiotics.


Subject(s)
Actinobacteria/genetics , Genome, Bacterial , Polyketide Synthases/genetics , Polymerase Chain Reaction/statistics & numerical data , Base Sequence , DNA Primers
9.
Microbiology (Reading) ; 151(Pt 1): 281-290, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15632445

ABSTRACT

The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912. LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure. Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE. Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription. Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass. In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.


Subject(s)
Aminoglycosides/biosynthesis , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Streptomyces/metabolism , Up-Regulation , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Microscopy, Confocal , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Streptomyces/genetics , Streptomyces/ultrastructure , Transcription, Genetic
10.
FEMS Microbiol Lett ; 222(1): 149-53, 2003 May 16.
Article in English | MEDLINE | ID: mdl-12757959

ABSTRACT

The regulatory genes lanI and lndI have been cloned from the landomycin A (LaA) producer Streptomyces cyanogenus S136 and from the landomycin E (LaE) producer Streptomyces globisporus 1912, respectively and both have been sequenced. A culture of S. globisporus I2-1 carrying a disrupted lndI gene did not produce LaE and other related intermediates. Complementation of S. globisporus I2-1 with either the lndI or lanI gene reconstituted LaE production indicating that LanI and LndI are involved in activation of structural genes in the respective clusters. Structural features of these regulatory genes are discussed.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents/biosynthesis , Streptomyces/genetics , Streptomyces/metabolism , Transcription Factors/genetics , 3' Flanking Region/genetics , 5' Flanking Region/genetics , Anti-Bacterial Agents/chemistry , Genes, Bacterial , Phenotype , Transcriptional Activation/genetics
11.
Antimicrob Agents Chemother ; 46(5): 1174-82, 2002 May.
Article in English | MEDLINE | ID: mdl-11959542

ABSTRACT

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.


Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Glycosides/biosynthesis , Streptomyces/metabolism , Bacterial Proteins/metabolism , Cloning, Molecular , Coumarins , Molecular Sequence Data , Multigene Family , Mutagenesis, Insertional , Open Reading Frames , Sequence Analysis, DNA , Streptomyces/genetics
12.
Microbiology (Reading) ; 148(Pt 2): 373-379, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11832501

ABSTRACT

Streptomyces viridochromogenes Tü57 is the principal producer of avilamycin A. aviG1, a putative methyltransferase gene, was detected in the avilamycin biosynthetic gene cluster. To determine the function of aviG1, a targeted gene inactivation experiment was performed. The resulting chromosomal mutant, carrying an in-frame deletion in aviG1, was deficient in avilamycin production. aviG1 was used to complement an eryBIII mutant of the erythromycin A producer Saccharopolyspora erythraea [Gaisser, S., Bohm, G. A., Doumith, M., Raynal, M. C., Dhillon, N., Cortes, J. & Leadlay, P. F. (1998). Mol Gen Genet 258, 78-88]. The presence of erythromycin A in the culture supernatant of the complemented mutant indicated that L-mycarose biosynthesis could be restored and that AviG1 could take over the function of the C-methyltransferase EryBIII.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Genes, Bacterial , Methyltransferases/genetics , Oligosaccharides/biosynthesis , Streptomyces/genetics , Streptomyces/metabolism , Amino Acid Sequence , Base Sequence , Carbohydrate Sequence , DNA, Bacterial/genetics , Erythromycin/biosynthesis , Gene Deletion , Genetic Complementation Test , Molecular Sequence Data , Molecular Structure , Mutation , Oligosaccharides/chemistry , Saccharopolyspora/genetics , Sequence Homology, Amino Acid
13.
Chem Biol ; 8(6): 557-67, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11410375

ABSTRACT

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.


Subject(s)
Anthraquinones/metabolism , Bacterial Proteins , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Amino Acid Sequence , Base Sequence , Catalytic Domain , Molecular Sequence Data , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Streptomyces/genetics , Substrate Specificity
14.
Chem Biol ; 8(6): 569-81, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11410376

ABSTRACT

BACKGROUND: Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents. RESULTS: A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins. CONCLUSIONS: The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacterial Proteins , Multigene Family , Oligosaccharides/biosynthesis , Streptomyces/genetics , Anti-Bacterial Agents/pharmacology , Cloning, Molecular , Gene Order , Genes, Regulator , Genetic Complementation Test , Methyltransferases/genetics , Methyltransferases/metabolism , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Oligosaccharides/pharmacology , Oxidoreductases/genetics , Oxidoreductases/metabolism , Parabens/metabolism , Polysaccharides/genetics , Polysaccharides/metabolism , Sequence Analysis , Streptomyces/metabolism
15.
Chem Biol ; 8(3): 253-63, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11306350

ABSTRACT

BACKGROUND: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS: The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS: The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.


Subject(s)
Anthraquinones/metabolism , Anti-Bacterial Agents/biosynthesis , Glycosyltransferases/genetics , Streptomyces/enzymology , Anti-Bacterial Agents/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Gene Library , Glycosyltransferases/chemistry , Molecular Sequence Data , Naphthacenes/metabolism , Sequence Alignment , Transformation, Genetic
16.
Plant Physiol ; 125(4): 1831-41, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11299363

ABSTRACT

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.


Subject(s)
Asteraceae/genetics , Asteraceae/metabolism , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Roots/metabolism , Transcription, Genetic , Amino Acid Sequence , Base Sequence , Darkness , Molecular Sequence Data , Naphthoquinones/metabolism , Plant Proteins/chemistry , RNA, Messenger/genetics , Restriction Mapping , Sequence Alignment , Sequence Homology, Amino Acid , Transformation, Genetic
17.
Antimicrob Agents Chemother ; 45(3): 690-5, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11181344

ABSTRACT

Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Anti-Bacterial Agents/pharmacology , Methyltransferases/genetics , Oligosaccharides/pharmacology , Streptomyces/drug effects , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/genetics , Chromatography, Affinity , Cloning, Molecular , Drug Resistance, Microbial/genetics , Drug Resistance, Microbial/physiology , Escherichia coli , Methyltransferases/metabolism , Microbial Sensitivity Tests , Molecular Sequence Data , Ribosomes/drug effects , Streptomyces/metabolism
18.
Chem Biol ; 8(12): 1239-52, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11755402

ABSTRACT

BACKGROUND: The genetic engineering of antibiotic-producing Streptomyces strains is an approach that became a successful methodology in developing new natural polyketide derivatives. Glycosyltransferases are important biosynthetic enzymes that link sugar moieties to aglycones, which often derive from polyketides. Biological activity is frequently generated along with this process. Here we report the use of glycosyltransferase genes isolated from the landomycin biosynthetic gene cluster to create hybrid landomycin/urdamycin oligosaccharide antibiotics. RESULTS: Production of several novel urdamycin derivatives by a mutant of Streptomyces fradiae Tü2717 has been achieved in a combinatorial biosynthetic approach using glycosyltransferase genes from the landomycin producer Streptomyces cyanogenus S136. For the generation of gene cassettes useful for combinatorial biosynthesis experiments new vectors named pMUNI, pMUNII and pMUNIII were constructed. These vectors facilitate the construction of gene combinations taking advantage of the compatible MunI and EcoRI restriction sites. CONCLUSIONS: The high-yielding production of novel oligosaccharide antibiotics using glycosyltransferase gene cassettes generated in a very convenient way proves that glycosyltransferases can be flexible towards the alcohol substrate. In addition, our results indicate that LanGT1 from S. cyanogenus S136 is a D-olivosyltransferase, whereas LanGT4 is a L-rhodinosyltransferase.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents/biosynthesis , Glycosyltransferases/genetics , Streptomyces/enzymology , Streptomyces/genetics , DNA Primers , Genes, Bacterial , Genetic Vectors , Glycosyltransferases/metabolism , Magnetic Resonance Spectroscopy , Multigene Family , Mutation , Sequence Analysis, DNA , Streptomyces/metabolism
19.
Chem Biol ; 7(11): 821-31, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11094336

ABSTRACT

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.


Subject(s)
Anthraquinones/metabolism , Antibiotics, Antineoplastic/biosynthesis , Deoxy Sugars/biosynthesis , Glycosyltransferases/genetics , Multigene Family/genetics , Streptomyces/genetics , Cloning, Molecular , Gene Silencing , Genetic Complementation Test , Glycosyltransferases/metabolism , Molecular Sequence Data , Molecular Structure , Mutation , Plasmids/genetics , Plasmids/metabolism , Streptomyces/enzymology , Substrate Specificity
20.
Microbiology (Reading) ; 146 ( Pt 1): 147-154, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10658661

ABSTRACT

Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3.35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZl, urdGT2, urdG and urdH) on an 8.05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose.


Subject(s)
Antibiotics, Antineoplastic/biosynthesis , Glycosyltransferases/metabolism , Oxygenases/metabolism , Streptomyces/enzymology , Anthraquinones/chemistry , Anthraquinones/metabolism , Blotting, Southern , Cosmids/genetics , Electrophoresis, Polyacrylamide Gel , Gene Deletion , Gene Library , Genetic Complementation Test , Glycosyltransferases/genetics , Molecular Sequence Data , Mutation , Open Reading Frames/genetics , Oxygenases/genetics , Restriction Mapping , Sequence Analysis, DNA , Streptomyces/genetics
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