Genetic engineering of glycolytic pathway by disrupting glyceraldehyde-3-phosphate dehydrogenase gene in an industrial strain of 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: 2013

Öğrenci: İBRAHİM SERTDEMİR

Danışman: GÜLAY ÖZCENGİZ

Özet:

Streptomyces clavuligerus is a gram-positive, filamentous bacterium which produces several important secondary metabolites, including isopenicillin N, cephamycin C and the β-lactamase inhibitor, clavulanic acid (CA). CA is being used in combination with commonly used β-lactam antibiotics in order to fight against bacterial infections that are resistant to such antibiotics. Glyceraldehyde-3-phosphate (GAP) which is an intermediate product of glycolytic pathway is also used in CA biosynthesis as a crucial substrate. In this study, S. clavuligerus gap1 encoding GAP dehydrogenase enzyme was targeted to canalize all produced GAP to CA biosynthetic pathway. In this way, a significant improvement of CA yield of an industrial strain of S. clavuligerus was aimed at. In the present study, gap1 amplified from S. clavuligerus genome was cloned into E. coli, and then disrupted by kanamycin resistance gene (aphII) cassette. The construct was cloned into the industrial strain; however, any gap1-disrupted mutant resulted from homologous recombination couldn’t be obtained following plasmid curing/selection process. However, for such metabolic engineering studies, a faster and more efficient plasmid curing technique was developed for S. clavuligerus which can be used for selection of disrupted mutants. For further study, after gap1::aphII mutant of S. clavuligerus is obtained, its CA yield will be compared with that in the parental strain through fermentation experiments.