Effect of homologous multiple copies of aspartokinase gene on cephamycin C biosynthesis in Streptomyces clavuligerus

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2005

Öğrenci: BİLGİN TAŞKIN

Danışman: GÜLAY ÖZCENGİZ

Özet:

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