Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis

2018 ◽  
Vol 102 (9) ◽  
pp. 4009-4023 ◽  
Author(s):  
Howard Ramirez-Malule ◽  
Stefan Junne ◽  
Mariano Nicolás Cruz-Bournazou ◽  
Peter Neubauer ◽  
Rigoberto Ríos-Estepa
Keyword(s):  
Clavulanic Acid ◽  
Strong Association ◽  
Tca Cycle ◽  
Streptomyces Clavuligerus ◽  
Acid Biosynthesis

scholarly journals In vivo functional analysis of a class A β-lactamase-related protein essential for clavulanic acid biosynthesis in Streptomyces clavuligerus

PLoS ONE ◽  
2019 ◽  
Vol 14 (4) ◽  
pp. e0215960 ◽  
Author(s):  
Santosh K. Srivastava ◽  
Kelcey S. King ◽  
Nader F. AbuSara ◽  
Chelsea J. Malayny ◽  
Brandon M. Piercey ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Clavulanic Acid ◽  
Streptomyces Clavuligerus ◽  
Related Protein ◽  
Acid Biosynthesis ◽  
Class A

scholarly journals Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

2004 ◽  
Vol 48 (1) ◽  
pp. 192-202 ◽  
Author(s):  
S. E. Jensen ◽  
A. S. Paradkar ◽  
R. H. Mosher ◽  
C. Anders ◽  
P. H. Beatty ◽  
...  
Keyword(s):  
Cell Wall ◽  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Production ◽  
Streptomyces Clavuligerus ◽  
Open Reading Frames ◽  
Acid Biosynthesis ◽  
Penicillin Binding Proteins ◽  
Total Absence ◽  
Reading Frames

ABSTRACT An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

scholarly journals Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

2000 ◽  
Vol 182 (14) ◽  
pp. 4087-4095 ◽  
Author(s):  
Rongfeng Li ◽  
Nusrat Khaleeli ◽  
Craig A. Townsend
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Streptomyces Clavuligerus ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Resistant Bacteria ◽  
Acid Biosynthesis ◽  
Significant Similarity ◽  
New Genes

ABSTRACT Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strainStreptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11(fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement andtrans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9(cad) in the clavulanic acid cluster. While theorf10 (cyp) and orf11(fd) proteins show homologies to other knownCYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.

scholarly journals Deletion of the pyc Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit

1999 ◽  
Vol 181 (22) ◽  
pp. 6922-6928 ◽  
Author(s):  
Rosario Pérez-Redondo ◽  
Antonio Rodríguez-García ◽  
Juan F. Martín ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Gene Product ◽  
Streptomyces Clavuligerus ◽  
Defined Medium ◽  
Thiamine Pyrophosphate ◽  
Acid Biosynthesis ◽  
Acid Protein ◽  
In The Wild ◽  
Lactamase Inhibitor ◽  
Amino Acid Protein

ABSTRACT The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster ofStreptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of thepyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with theaph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing d-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. Thepyc replacement mutant overproduces cephamycin.

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

2004 ◽  
Vol 186 (19) ◽  
pp. 6501-6507 ◽  
Author(s):  
A. de la Fuente ◽  
J. F. Martín ◽  
A. Rodríguez-García ◽  
P. Liras
Keyword(s):  
Clavulanic Acid ◽  
Streptomyces Clavuligerus ◽  
Culture Conditions ◽  
Transferase Activity ◽  
Reaction Products ◽  
Acid Biosynthesis ◽  
Substrate Specificities ◽  
Acetyltransferase Activity ◽  
Standard Culture

ABSTRACT 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.

Cloning A Streptomyces clavuligerus Genetic Locus Involved in Clavulanic Acid Biosynthesis

1984 ◽  
Vol 2 (9) ◽  
pp. 808-811 ◽  
Author(s):  
Christopher R. Bailey ◽  
Michael J. Butler ◽  
Ian D. Normansell ◽  
Robert T. Rowlands ◽  
David J. Winstanley
Keyword(s):  
Clavulanic Acid ◽  
Genetic Locus ◽  
Streptomyces Clavuligerus ◽  
Acid Biosynthesis

scholarly journals Streptomyces clavuligerus Has a Second Copy of the Proclavaminate Amidinohydrolase Gene

2004 ◽  
Vol 48 (2) ◽  
pp. 514-520 ◽  
Author(s):  
Susan E. Jensen ◽  
Annie Wong ◽  
Alison Griffin ◽  
Barry Barton
Keyword(s):  
Clavulanic Acid ◽  
Streptomyces Clavuligerus ◽  
Its Sequence ◽  
Acid Biosynthesis ◽  
Cross Hybridization ◽  
Genetic Studies ◽  
Defective Mutant ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Dna Fragment

ABSTRACT Past genetic studies have indicated that the genes encoding early enzymes of clavulanic acid biosynthesis may be duplicated in Streptomyces clavuligerus. We observed cross-hybridizing bands upon Southern analyses of proclavaminate amidinohydrolase (pah)-defective mutant strains of S. clavuligerus screened with a pah-specific probe. The DNA fragment responsible for this cross hybridization was cloned and sequenced and shown to encode a second copy of the pah gene. The new pah gene (pah1) was 1,056 bp in length, and its sequence was 72% identical to that of the original pah gene (pah2). Disruption mutants with defects in pah1 showed no significant effects on production of clavulanic acid or any of the clavam metabolites with stereochemistries opposite that of clavulanic acid (5S clavams) produced by S. clavuligerus when they were grown on starch asparagine or soy medium. However, double mutants with defects in both pah1 and pah2 were defective in the production of both clavulanic acid and all of the 5S clavam metabolites.

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