利用同框敲除技术研究Ⅲ型聚酮合酶基因orf18的代谢产物

Studying the metabolites catalyzed by the type Ⅲ polyketide synthase gene orf18 through in-frame deletion

为了阐明Streptomyces sahachiroi ATCC 33158中Ⅲ型聚酮合酶基因orf18的功能,利用同源双交换的方法对该基因进行同框敲除,采用异位表达的方法进行了相应的回补实验。通过高效液相色谱及液相色谱-质谱联用分析发现基因orf18的敲除会明显降低发酵产物中苯甲酸盐的含量,回补该基因能够在一定程度上恢复苯甲酸盐的产量,表明orf18在原宿主中极有可能参与苯甲酸盐的生物合成。

To verify the function of orf18,a typeⅢ polyketide synthase gene isolated fromStreptomyces sahachiroi ATCC 33158 was used to construct the knockout mutant strain by in-fame deletion of orf18 and the complementation mutant strain by ectopic expression of orf18 in the knockout strain.The results of HPLC and LC-MS analyses showed that knockout of orf18 significantly reduced the benzoic acid production.Complementation of orf18 recovered the yield of benzoic acid.It is indicated that orf18 is involved in the biosynthesis of benzoic acid in the original host strain.