棒状链霉菌中lat基因的突变及其对克拉维酸产量的影响

以棒状链霉菌Streptomyces clavuligerus NRRL3585的染色体为模板扩增得到1条1.77kb的lat基因片段,并将其用于构建重组质粒pKCLES。将pKCLES由E.coli ET12567接合转移至S.clavuligerus中。重组质粒pKCLES与S.clavuligerus染色体中野生型lat基因发生同源交换,从而获得了带有阿泊拉抗性的突变菌株。对S.clavuligerus NRRL3585以及lat突变菌株的基因组进行PCR验证,且测定了这2种菌株的产头酶素C的能力。结果均表明,突变菌株中的lat基因中插入了含有阿泊拉抗性的pKC1139片段而遭到了破坏。另外,以HPLC法测定了这2种菌株在不同培养时间(72h和96h)下的克拉维酸的产量,结果显示,lat突变菌株的克拉维酸产量最高能达到其原始菌株的2.3倍。

The Mutation of lat Gene in Streptomyces clavuligerus and Its Effect on the Production of Clavulanic Acid

A 1.77 kb fragment of lat gene was obtained by PCR from Streptomyces clavuligerus NRRL 3585 chromosome DNA.Recombinant plasmid pKCLES,which was used to disrupt the lat gene,was constructed.pKCLES was introduced into S.clavuligerus by intergeneric conjugation from E.coli ET12567.The lat disrupted transformants were obtained through homogeneous cross-over between the recombinant plasmid pKCLES and the S.clavuligerus chromosome.The clavulanic acid yield in the wild type strain and the mutant was analyzed by HPLC in different incubation time intervals(72h and 96h).The results showed that the clavulanic acid yield in the lat mutant was approximately 2.3 fold higher than the wild type strain at their highest production point.