Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system

Pelzer, Stefan; Reichert, Wolfgang; Huppert, Michaela; Heckmann, D.; Wohlleben, Wolfgang

doi:10.1016/s0168-1656(97)00082-5

Cited by 74 publications

(65 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1). For transformation of A. mediterranei DSM5908, a modified direct transformation method was used as described previously (6). After transformation, the clones harboring the erythromycin selection marker had integrated the pIFCS plasmid over a single crossover into the chromosome.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Polyketide Synthase in Glycopeptide Biosynthesis

Pfeifer¹,

Nicholson²,

Ries³

et al. 2001

Journal of Biological Chemistry

Self Cite

144

View full text Add to dashboard Cite

Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20 -30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Recently, the gene cluster for balhimycin (4) biosynthesis has been identified in Amycolatopsis mediterranei DSM5908 by gene inactivation experiments (5), and the previously established genetic system (6) now permits the precise identification of gene functions and also the construction of new strains able to produce modified glycopeptides (5,(7)(8)(9).…”

mentioning

confidence: 99%

A Polyketide Synthase in Glycopeptide Biosynthesis

Pfeifer¹,

Nicholson²,

Ries³

et al. 2001

Journal of Biological Chemistry

Self Cite

144

View full text Add to dashboard Cite

show abstract

“…E. coli strains were grown under standard conditions. pBluescript SKk (pSKk) was obtained from Stratagene and plasmid pSP1, carrying the erythromycin-resistance gene (Pelzer et al, 1997), was provided by S. Pelzer, Department of Microbiology, University of Tu$ bingen. pKC1132, carrying the apramycin-resistance gene, was a gift from Eli Lilly.…”

Section: Methodsmentioning

confidence: 99%

Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae Tü2717 This paper is dedicated to Professor Heinz Floss, a pioneer in the field of antibiotics, on the occasion of his 65th birthday. The GenBank accession numbers for the sequences reported in this paper are AF164960 and AF164961.

et al. 2000

View full text Add to dashboard Cite

Urdamycin A, the principal product of Streptomyces fradiae Tu $ 2717, 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 335 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) on an 805 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.

show abstract

“…(5-7), and Amycolatopsis sp. (8,9), synthesis of a phytoionophore in Pseudomonas syringae (10), and synthesis of the immunosuppressant cyclosporin A in the fungus Tolypocladium niveum (11). Unusual amino acids could also be incorporated into the ribosomally translated core protein through post-translational modifications, however.…”

mentioning

confidence: 99%

Presence of D-Alanine in an Endopeptidase from Streptococcus pyogenes

Lee

Fischetti

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

D-Amino acids are commonly found in peptide antibiotics and the cell wall peptidoglycan of bacterial cell walls but have not been identified in proteins or enzymes. Here we report the presence of 6 -7 D-alanine residues in an endopeptidase of Streptococcus pyogenes, a unique enzyme involved in surface protein attachment that we term LPXTGase. Using D-amino acid oxidase coupled with catalase for the deamination of D-alanine to pyruvic acid (a conversion unique to D-alanine), we were able to identify [ 14 C]pyruvic acid in a [ 14 C]alaninelabeled preparation of purified LPXTGase, which represents 27% of the amino acid composition. Because Damino acids are not accommodated in ribosomal peptide synthesis, these results suggest that the same process used in assembling peptide antibiotics or a yet unidentified mechanism may synthesize the core protein of this endopeptidase.In our previous report we described the unusual properties of an endopeptidase termed LPXTGase purified from Streptococcus pyogenes (1). This membrane-associated enzyme cleaves a common sequence motif, LPXTG, found near the C termini of precursor proteins programmed to be displayed on the cell surface and linked to the cell wall peptidoglycan. We discovered that the LPXTGase is highly glycosylated and contains hydrophobic amino acids with unusually high masses, which are not among the 20 common amino acids present in ribosomally translated proteins. The presence of unusual amino acids indicated the possibility that the core protein of the enzyme might be constructed entirely or in part through the well characterized nonribosomal peptide synthesis pathway, where amino acids are assembled into a peptide on amino acid-activating multienzyme templates. Examples are the syntheses of peptide antibiotics in Bacilli sp. (2-4), Streptomyces sp. (5-7), and Amycolatopsis sp. (8, 9), synthesis of a phytoionophore in Pseudomonas syringae (10), and synthesis of the immunosuppressant cyclosporin A in the fungus Tolypocladium niveum (11). Unusual amino acids could also be incorporated into the ribosomally translated core protein through post-translational modifications, however. Another unusual feature of the core protein of LPXTGase is that only seven different amino acids represent 93% of the 61 common residues present in the core protein. It is particularly striking that alanine accounts for nearly 40% of the common amino acids found in the protein.Over-representation of one or two amino acids within a peptide is a characteristic feature of nonribosomally constructed peptide antibiotics (4, 5). As there exists an intriguing possibility that the core protein is indeed constructed at least in part by a nonribosomal peptide synthesis pathway, we considered whether some of the alanine residues might be in the D-form, a hallmark feature of nonribosomal synthesis of peptide antibiotics (11,12). This prompted us to search for the presence of D-alanine in the core protein of LPXTGase. In this report, we present strong evidence that alanine in the D-isoform is present in...

show abstract

Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system

Cited by 74 publications

References 37 publications

A Polyketide Synthase in Glycopeptide Biosynthesis

A Polyketide Synthase in Glycopeptide Biosynthesis

Presence of D-Alanine in an Endopeptidase from Streptococcus pyogenes

Contact Info

Product

Resources

About