棒状链霉菌中lat基因的置换与克拉维酸的选择性生产

Selective production of clavulanic acid by lat gene replacement in Streptomyces clavuligerus

目的提高棒状链霉菌的克拉维酸生产能力 ,去除副产物头霉素C。方法将安普霉素抗性基因片段插入到 4 7kb的lat pcbAB基因片段中 ,采用接合转移方法对棒状链霉菌中头霉素C生物合成的关键基因lat基因进行了置换。结果与结论经PCR验证 ,染色体上正常的lat pcbAB基因被插入失活的基因所替代。对重组菌株的发酵产物进行HPLC检测 ,发现所有的重组菌株均不再合成头霉素C ,并且重组菌株C35 85 lat ::apr、C2 5 1 lat::apr和C16 6 lat::apr的克拉维酸产量较出发菌株分别提高了 2 6 7 6 8%、4 3 6 9%和 35 0 5 %。结果表明基因插入失活是去除多余组分 。

Objective To improve the production of clavulanic acid and to genetically delete cephamycin C in the fermentation of Streptomyces clavuligerus.Methods A 1.5 kb apramycin resistance gene fragment was inserted into the 4.7 kb latpcbAB gene (the key genes for the biosynthesis of cephamycin C )fragment,then was introduced into Streptomyces clavuligerus by conjugal transfer.Double cross-over strains were obtained by antibiotic-resistance selection.Results and conclusion Gene replacement is confirmed by the analysis of PCR products.The HPLC analysis of fermentation products shows that all the resulting recombinant strains have stopped cephamycin C production,while the yield of clavulanic acid has elevated 267.68%,43.69% and 35.05% respectively in the recombinant strains C3585-lat::apr,C251-lat::apr and C166-lat::apr.Above results have demonstrated that gene replacement is a possible way in deleting unwanted components and in increasing the yield of useful components of microorganisms.