Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus


Yilmaz E. I. , Caydasi A. K. , ÖZCENGİZ G.

JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, cilt.35, ss.1-7, 2008 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 35 Konu: 1
  • Basım Tarihi: 2008
  • Doi Numarası: 10.1007/s10295-007-0259-8
  • Dergi Adı: JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
  • Sayfa Sayıları: ss.1-7

Özet

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.