Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus |
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Authors: | Ebru I Y?lmaz Ayse K Çaydasi Gülay Özcengiz |
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Institution: | (1) Biology Department, Middle East Technical University, Ankara, 06531, Turkey;(2) Biology Department, Dicle University, Diyarbakır, 21280, Turkey |
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Abstract: | The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for β-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M
r of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and
in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free l-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium. |
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Keywords: | Streptomyces clavuligerus Homoserine dehydrogenase Cephamycin C Aspartate pathway Gene disruption |
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