Cloning and characterization of Streptomyces clavuligerus meso-diaminopimelate decarboxylase (lysA) gene

Yağcıoğlu, Çiğdem
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-diaminopimelic acid (DAP) decarboxylase (E.C. catalyzing the conversion of diaminopimelate to lysine was cloned and characterized for the first time from S. clavuligerus NRRL 3585. The attempts to clone the gene by constructing libraries of S. clavuligerus genomic DNA and screening of the libraries either by homologous probing or complementation approach gave no positive results. Then, PCR-based cloning was taken as the approach and the gene was amplified with PCR using the primers derived from the conserved sequences of lysA genes in two fragments (620 and 983 bp) which had overlapping regions. Fragments were then cloned and nucleotide sequencing revealed a complete open reading frame (ORF) encoding a protein of 463 aa (Mr 49, 907). The GC content of the gene was identified as 70.98 %. The gene sequence showed 83 % identity to the sequence of S. coelicolor lysA gene and 81 % identity to S. avermitilis lysA gene. By comparing the amino acid sequence of this protein to those available in database, the sites of the enzyme important for catalysis were identified.


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Taşkın, Bilgin; Özcengiz, Gülay; Department of Biotechnology (2005)
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 ?-amin...
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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. ...
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Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum P-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete...
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Citation Formats
Ç. Yağcıoğlu, “Cloning and characterization of Streptomyces clavuligerus meso-diaminopimelate decarboxylase (lysA) gene,” M.S. - Master of Science, Middle East Technical University, 2004.