Determination Of Clavulanic Acid And Cloning And Mutation Of The Lat Gene Of Streptomyces Clavuligerus

Clavulanic acid is the earliest β-lactamase inhibitor used in clinic. It is produced by Streptomyces clavuligerus which has been well characterized for its production of lactam compounds. Clavulanic acid is very important both clinically and economically for its wide range β-lactamase inhibitor activity.In this study, clavulanic acid-producing strain Streptomyces clavuligerus was studied. Clavulanic acid in fermentation liquor of Streptomyces clavuligerus was assayed by HPLC, spectrophotometry and bioassay. And the relationship between the results determined by three methods were investigated. Conclusion shows that results determined by three methods were correlation.lat gene of the clavulanic acid gene cluster which encoding lysine- ε -amino tranferase in Streptomyces clavuligerus was altered. Plasmid pKCLES and pKCLHS which were the inactivated lat gene fragment was introduced into MCS of E.coli-Streptomyces shuttle vector pKC1139 (containing oriT gene) were constructed in E.coliDH5. pKCLHS was transformed into Streptomyces clavuligerus and lat mutant was obtained.In order to obtain lat gene fragment, primers were designed and synthesized. A 1.8 kb lat gene fragment was obtained by PCR with genomic DNA of Streptomyces clavuligerus as a template. This fragment was inserted into the MCS of pUCm-T vector and recombinant plasmid pUCm-T-lat was obtained. After digested pUCm-T-lat with EcoRI-Hind III, the lat gene fragment was subcloned to E.coli-Streptomyces shuttle vector pKC1139 to give recombinant plasmid pKC1139-lat. pKC1139-lat was digested to eliminate part of lat gene. The remaining parts were self-ligated to give recombinant plasmid pKCLES and pKCLHS.Parameters affecting protoplast formation and regeneration of Streptomyces clavuligerus were studied in detail. pKCLHS was introduced into Streptomyces clavuligerus by PEG1000 assisted protoplast transformation. Through homogeneous single cross-over lat mutant strains were obtained.