Application Of Gene Disruption And Gene Expression To Streptomyces In The Improvement Of Components Of Antibiotics

The fermentation products of streptomyces are multi-component because of its complex second metabolism. However, only a few of the components are bioactive. Application of gene disruption and gene expression to streptomyces will eliminate undesirable metabolic products or improve the yield of useful components.In this article, a 1.5 kb apramycin resistance gene replaced the 0.7 kb inner fragment of lat-pcbAB genes (the key genes for the biosynthesis of cephamycin C in Streptomyces clavuligerus) to make the disruption plasmid pSY06.Then pSY06 was introduced into Streptomyces clavuligerus by conjugal transfer. Double cross-over strains were obtained by antibiotic-resistance selection. The HPLC analysis of fermentation products showed that resulting recombinant strains having opposite direction of transcription between Am and lat-pcbAB genes stopped cephamycin C production, while the yield of clavulanic acid was elevated in different degrees.Placing aveC gene in the downstream of thiostrepton-induced promotor Ptip_A obtained the recombinant plasmid pSY14, then pSY14 was introduced into Streptomyces avermitilis C9-aveC::Am that aveC gene had been disrupted by conjugal transfer. Results showed that aveC gene was expressed by the induction of both thiostrepton and nosiheptide. Recombinant strains of C-9 restored the ability to synthesis "1" component of avermectins.The bkdF gene fragment of Streptomyces avermitilis was inactivated by inserting a 1.5 kb apramycin resistance gene fragment in opposite orientation. The recombinant plasmid pSY25 was introduced into Streptomyces avermitilis M2 by conjugal transfer. The HPLC analysis of fermentation products showed that the ability of synthesising avermectins was decreased significantly.