Emerging evidence for a shared biosynthetic pathway among clavulanic acid and the structurally diverse clavam metabolites

Janc, James W.; Egan, Laura A.; Townsend, Craig A.

doi:10.1016/s0960-894x(01)80946-x

Cited by 18 publications

(15 citation statements)

References 13 publications

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“…However, the clavulanic acid production by the S. clavuligerus oat2 mutant in the presence of high arginine concentrations increased relative to that of the wild-type strain, at some times reaching the levels of the wild type in the absence of arginine. Similar results were observed for the production of alanylclavam, a branching metabolite of the clavulanic acid pathway (10). These results suggest that an Oat2 reaction product (or the protein itself) has a negative regulatory role on clavulanic acid at high arginine concentrations that is removed in the mutant with the oat2 deletion.…”

Section: Discussionsupporting

confidence: 79%

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

et al. 2004

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The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.Streptomyces clavuligerus produces clavulanic acid (CA), an important ␤-lactamase inhibitor synthesized by the condensation of arginine (23,27), and glyceraldehyde-3-phosphate to form carboxyethylarginine (12, 18). In wild-type strains, arginine is a limiting precursor for clavulanic acid biosynthesis (23). Studies of the arginine gene cluster have been highly interesting as a way of understanding the channeling of this precursor into clavulanic acid (20,21).The presence of a cyclic arginine pathway in Streptomyces species has been reported previously (20). The S. clavuligerus arginine gene cluster contains an argJ gene encoding a protein of 39,733 Da, similar to other ArgJ proteins, as deduced from the genome sequences of Streptomyces coelicolor (74.9% identical amino acids), Mycobacterium tuberculosis (47.8% identity), and Corynebacterium glutamicum (40.7% identity). ArgJ recycles the acetyl group from N-acetylornithine to L-glutamic acid in the first step of the pathway. The argJ gene complements an Escherichia coli XSD2 mutant lacking N-acetylornithinase activity, indicating that the ornithine N-acetyltransferase (OAT) activity encoded by argJ is able to complement the N-acetylornithinase activity encoded by the argE gene of the lineal arginine pathway in E. coli.In the clavulanic acid gene cluster of S. clavuligerus, the oat2 gene (initially described as ORF6), located downstream of the pah gene, encodes a protein of 41,607 Da with a high similarity to ornithine acetyltransferases (9). ArgJ and Oat2 share 31.1% amino acid identity over the entire sequence. When oat2 was subcloned and transformed into E. coli XSD2, the argE mutation of this strain was complemented, indicating that the oat2 promoter was expressed in E. coli; moreover, the transformants showed a strong OAT activity but no ornithinase activity (20). A putative ARG box, which is characteristic of genes tha...

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Section: Discussionsupporting

confidence: 79%

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

et al. 2004

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“…tsp, hybridization signal due to protection by the shorter transcript. (19,20), the two CAS isoenzymes may simply function to provide enough precursors to be channeled into the two pathways. Since alanylclavam was detected only in soy medium-grown cultures, in which both cas1 and cas2 are expressed, and not in SA medium, in which only cas2 is expressed, this implies either that cas1 is specifically required for clavam production and cas2 cannot substitute or, more probably, that some as yet unidentified activity(ies) specific for the clavam branch of the pathway has the same type of nutritional regulation as does CAS1.…”

Section: Discussionmentioning

confidence: 99%

“…The latter conversion is thought to involve several steps, including a stereochemical conformational change in the bicyclic ring structure, but these steps have not yet been fully characterized (6,36). Recent experiments with labelled substrates have shown that proclavaminic acid can be incorporated into both clavulanic acid and the other clavam metabolites (19,20). The biosyntheses of all clavam metabolites, including clavulanic acid, are therefore thought to follow a common pathway, at least up to proclavaminic acid and perhaps up to clavaminic acid.…”

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confidence: 99%

Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus

1995

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A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon.

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“…It has a typical ␤-lactam structure characterized by a bicyclic nucleus comprising a four-member ␤-lactam ring fused to a five-member oxazolidine ring (21, 48), but despite its structural similarity to the conventional ␤-lactam antibiotics, such as penicillins and cephalosporins, which are synthesized by a nonribosomal peptide synthetase mechanism, CA utilizes a carboxyethylarginine synthase (CeaS)-catalyzed reaction for the initial condensation of precursors: glyceraldehyde-3-phosphate (G3P) and arginine (14,27,51,62). CA and 5S clavams share an early biosynthetic pathway that gives rise to the common intermediate clavaminic acid (13,22). Earlier genetic studies on the genes involved in CA and 5S clavam biosynthesis revealed one of the most complicated examples of gene organization governing antibiotic biosynthesis, showing that the genetic information needed for the production of CA and the 5S clavams is distributed among three distinct gene clusters located in unlinked regions of the S. clavuligerus genome (54, 60).…”

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confidence: 99%

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

Yin¹,

Xiang²,

Zheng³

et al. 2012

Appl Environ Microbiol

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ABSTRACTIn bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption ofargRinStreptomyces clavuligerusNP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and itsargRdisruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin inS. clavuligerus.

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Emerging evidence for a shared biosynthetic pathway among clavulanic acid and the structurally diverse clavam metabolites

Cited by 18 publications

References 13 publications

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

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