Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus.
The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for beta-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.
Yilmaz
Ebru I
EI
Biology Department, Middle East Technical University, Ankara, 06531, Turkey.
Caydasi
Ayse K
AK
Ozcengiz
Gülay
G
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D002513:Cephamycins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing; D006715:Homoserine Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002513:Cephamycins; D003470:Culture Media; D011994:Recombinant Proteins; D006715:Homoserine Dehydrogenase; C024925:cephamycin C