Effect of homologous multiple copies of aspartokinase gene on cephamycin c biosynthesis in streptomyces clavuligerus

Taşkın, Bilgin
Streptomyces clavuligerus is a gram-positive filamentous bacterium well known for its ability to produce an array of ?-lactam compounds (secondary metabolites) including cephamycin C, clavulanic acid and other structurally related clavams. Of these, cephamycin C is a second generation cephalosporin antibiotic having great medical significance. Biosynthesis of the β-lactam nucleus begins with the non-ribosomal condensation of L-?-aminoadipic acid (?-AAA), L-cysteine and L-valine to form the tripeptide ?-aminoadipiyl-cysteinyl-valine (ACV). In Streptomyces clavuligerus, ?-aminoadipic acid (?-AAA) is a catabolic product of L-lysine produced from the lysine branch of the aspartate pathway and its biosynthesis represents a key secondary metabolic regulatory step in carbon flow to β-lactam synthesis through this core pathway. The ask (aspartokinase)-asd (aspartate semialdehyde dehydrogenase) gene cluster which encodes for the first key enzymes of aspartate pathway has already been cloned from S. clavuligerus, characterized and heterologously expressed for the first time in our laboratory. Amplification of ask-asd cluster or ask gene alone in a multi-copy Streptomyces plasmid vector and determination of the effects of multiple copies on cephamycin C biosynthesis were the goals of the present study. For this purpose, three different strategies were employed. Of these, two strategies involving the use of vector pIJ702 did not work because of the instability of resulting recombinant plasmids. In the third and last strategy, we used another multicopy Streptomyces vector, pIJ486, which we showed in this study to be very stable for the same purpose. Meanwhile, an efficient protoplast transformation protocol was developed in our laboratory. Ask gene was cloned into this vector and S. clavuligerus protoplasts were efficiently transformed with the recombinant plasmid (pTB486) using


Cloning and characterization of Streptomyces clavuligerus meso-diaminopimelate decarboxylase (lysA) gene
Yağcıoğlu, Çiğdem; Özcengiz, Gülay; Department of Biology (2004)
In Streptomyces clavuligerus, the route to the biosynthesis of a-aminoadipic acid (a-AAA) represents an important primary metabolic pathway providing carbon flux to the synthetases of antibiotic formation. This carbon flow comes through the lysine-specific branch of the aspartate pathway and is rate limiting in the formation of cephamycin C, a second generation cephalosporin produced by this organism. In this study, the lysA gene which encodes for an important key enzyme of aspartate pathway; meso-diaminopi...
Identification of streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis from sputum samples of patients with community acquired pneumonia by polymerase chain reaction
Güçlü, Aylin Üsküdar; Gözen, Ayşe Gül; Department of Biology (2005)
The present work describes the evaluation of the value of polymerase chain reaction in diagnosis of pneumonia caused by the most common three bacterial pathogens; Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis from sputum of patients with community acquired pneumonia admitted to The Department of Pulmonary Diseases of Gulhane Military Medical Academy. In this study, 107 sputa from 142 patients with suspected community acquired pneumonia were used to survey the causative agents. ...
Characterization and purification of a bacteriocin produced by Leuconostoc mesenteroides subsp. cremoris
Dündar, Halil; Bozoğlu, Faruk; Department of Biotechnology (2006)
In this study, a new bacteriocin isolated from a Leuconostoc mesenteroides subsp. cremoris strain was purified to homogeneity by pH mediated cell adsorption-desorption, solid phase extraction with Amberlite XAD-16, cation-exchange chromatography and hydrophobic interaction chromatography. The purification resulted in an electrophoretically pure protein that was smaller than 6 kDa as judged by SDS-PAGE. The bacteriocin was found to be very hydrophobic and cationic and could be adsorbed by synthetic calcium s...
Targeted disruption of homoserine dehydrogenase gene in Streptomyces clavuligerus and its effects on cephamycin C production
Çaydaşı (Koca), Ayşe; Özcengiz, Gülay; Department of Biology (2006)
The members of the genus Streptomyces are well-known for their capacity to synthesize a vast repertoire of secondary metabolites, including many useful antibiotics and proteins. Streptomyces clavuligerus is the producer of the medically important β-lactam antibiotics such as cephamycin C and the potent β-lactamase inhibitor clavulanic acid. The aspartate pathway of S. clavuligerus is an important primary metabolic pathway providing substrates for β-lactam synthesis. This pathway uses L-aspartic acid as the ...
Identification of serotype specific dna marker for salmonella typhimurium by rapd-pcr method
Aksoy, Ceren; Gültekin, Güzin Candan; Department of Biotechnology (2004)
This study was performed for the identification of specific DNA marker using RAPD-PCR (Random Amplified Polymorphic DNA) method for serotype Salmonella Typhimurium which is one of the most prevalent serotype causing food poisining all over the world. The Primer 3 (RAPD 9.1), 5̕-CGT GCA CGC-3ء, was used in RAPD-PCR with 35 different Salmonella isolates. 12 of them were serotype Salmonella Typhimurium and 23 of them were belonging to other six different serotypes. Accordingly, two different 300 bp and 700 bp ...
Citation Formats
B. Taşkın, “Effect of homologous multiple copies of aspartokinase gene on cephamycin c biosynthesis in streptomyces clavuligerus,” M.S. - Master of Science, Middle East Technical University, 2005.