| Cephamycin C produced by Streptomyces clavuligerus is a kind ofβ-lactam antibiotics.Extensive attention has focused on cephamycin C because of its good stability againstβ-lactamases. The fermentation products of Streptomyces clavuligerus includes a few kind ofβ-lactam compounds such as cephamycin C and clavulanic acid. The aim of this paper is to construct a gene-engineering strain, which produces cephamycin C only, by the disruption of the biosynthesis gene of clavulanic acid though the methods of gene-engineering.Firstly, the recombinant plasmid pBH14 was obtained by inserting the apramycin resistance gene into the pyc gene(one of the key genes in the biosynthesis of clavulanic acid) fragment of streptomyces clavuligerus. The recombinant plasmid was introduced into S.clavuligerus NRRL3585 by conjugal transfer and the recombinant strains NRRL3585-B1 was obtained .The HPLC analysis of fermentation products produced by recombinant strains showed that the content of clavulanic acid decreased obviously . However, the content of cephamycin C was also decreased.Secondly, a point mutation was introduced into pyc gene fragment by PCR amplification. The point mutation plasmid pBH25 was introduced into NRRL3585-B1 by conjugal transfer. The HPLC analysis of fermentation products produced by recombinant strains showed that the content of clavulanic acid descends while the yield of cephamycin C was elevated in different degrees. But the genetic stability of recombinant strains was not ideality.Then, a deletion mutation, deleting fifteen amino acids, was introduced into pyc gene fragment by PCR amplification. The deletion mutation plasmid pBH35 was introduced into NRRL3585-B1 by conjugal transfer. HPLC analysis of fermentation products produced by recombinant strains showed that the content of clavulanic acid descend while the yield of cephamycin C was elevated. The genetic characters of recombinant strains is stable after several generation of culture.
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