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利用成功构建的基因缺失载体pLJ04(pKC1139∷△bkdF+△bkdH)对阿维菌素(avermectin)高产菌阿维链霉菌(Streptomyces avermitilis)76-02-e的bkdFGH基因进行缺失,获得的bkdFGH缺失突变株经过摇瓶发酵和HPLC检测,发现该突变株完全丧失了产生阿维菌素的能力。2-甲基丁酸及异丁酸的前体添加试验表明,当有外源前体存在时,突变株又能恢复阿维菌素合成的能力。将该bkdFGH基因缺失突变株命名为S.avermitilisbkd76-3。环己羧酸(CHC)前体添加试验及HPLC检测发现存在4种产物,经LC/MS分析验证,其中两种产物分别为CHC-B1和CHC-A2。
The deletion of the bkdFGH gene of Streptomyces avermitilis 76-02-e, avermectin-producing strain, was successfully constructed by using the gene deletion vector pLJ04 (pKC1139 :: △ bkdF + △ bkdH) Strains were shake flask fermentation and HPLC detection and found that the mutant completely lost the ability to produce avermectin. The addition of 2-methylbutyrate and isobutyrate precursors showed that the mutants restored the ability of abamectin to synthesize when exogenous precursors were present. The bkdFGH gene deletion mutant was named S.avermitilisbkd76-3. Cyclic carboxylic acid (CHC) precursor addition test and HPLC test found that there are four products, confirmed by LC / MS analysis, two of which were CHC-B1 and CHC-A2.