【作者】 陈光；

【导师】 何建勇；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2006， 硕士

【摘要】 阿维菌素（Avermectin）是由阿维链霉菌（Streptomyces avermitilis）产生的一类结构相似的十六元大环内酯齐墩果糖双糖衍生物，阿维菌素具有广谱、高效的抗寄生虫活性，对宿主不良反应极低，是目前理想的驱虫、杀虫药物。但阿维链霉菌在发酵过程中产生8个阿维菌素组分，其中只有B_1a组分的活性最高，因此，如何去除无用组分，提高有效组分B_1a的含量已成为世界各国竞相研究的热点。 本论文探索性地对影响阿维菌素组分的关键基因aveC基因和aveD基因进行了基因改造。 aveC基因编码阿维菌素生物合成的脱水酶，提高该基因的表达量可以提高有效组分“1”的比例。本论文采用基因工程的方法在阿维链霉菌的基因组DNA中增加了一套aveC基因拷贝，期望通过增加基因的拷贝数增强脱水酶的活性。对重组菌株PCR检验的结果表明，第二套aveC基因正确地插入到了基因组DNA的预定部位。然而，对重组菌株发酵产物的HPLC分析显示，阿维菌素“1”组分含量并没有明显的变化，提示该脱水酶的活性可能不是阿维菌素生物合成的限制因素。 aveD基因是编码阿维菌素C₅-O-甲基转移酶的基因，该酶催化阿维菌素“B”组分转化成为无用的“A”组分。为了去除无用的产物“A”组分，本论文采用了插入抗性基因的方法构建了aveD基因失活的重组质粒pCG23，并将该质粒转入阿维链霉菌AVD2-14中，得到重组菌株AV-C337。发酵结果显示重组菌株AV-C337仅产生阿维菌素B_2a和B_1a两个组分，彻底去除了无用的“A”组分，但重组菌株的产量较低。 为了避免插入较大外源基因可能造成的产量降低，本论文还采用了PCR扩增的方法，在aveD基因中引入突变，删除了aveD基因中5个保守的氨基酸序列，得到重组质粒pCG29。将其转入阿维链霉菌AV-C337中，通过同源双交换得到了缺失5个氨基酸的aveD基因失活阻断变株AV-C519。然而发酵结果表明，该阻断变株阿维菌素的产量也显著降低。因此，如何避免基因插入失活后代谢产物产量的降低，依然是个需要探索和解决的问题。更多还原

【Abstract】 Avermectins are 16-membered macrocyclic polyketides produced by Streptomyces avermitilis. They are known to be excellent anthelmintic agents with a good potency and broad spectrum of activity against a variety of nematode and anthropod parasites, and with a low level of side-effects on the host. Eight compounds are produced by S. avermitilis. Among these compounds, the B1a compound exhibits the most potent activity. Therefore, the research of eliminating undesirable metabolic products and enhancing the yield of useful component B1a is becoming a hotspot among many countries.In this study, aveC and aveD which were considered to be the key genes of avermectins biosynthesis, were altered by the method of gene engineering for the purpose of exploring.aveC encodes a dehydratase, and the high level of its expression could potentially enhance the ratio of "1" component to other components. In this paper, another aveC copy was inserted into the genomic DNA of S. avermitilis by gene manipulation, expecting to enhance the activity of dehydratase. By PCR amplification of the genomic DNA of recombinant strains, it was testified that the second aveC copy was inserted exactly into the expected site. However, the HPLC analysis of fermentation products produced by recombinant strains showed that the content of "1" component changed indistinctively. It implied that the activity of dehydratase may not be the limiting factor for avermectin biosythesis in S. avermitilis.aveD encodes a C₅-O-methyltransferase, which catalyze the coversion of "B" component to an useless "A" component. To eliminate "A" component, a disruption plasmid pCG23 was constructed by inserting an apramycin resistance gene into aveD gene fragment. Then the plasmid was introduced into S. avermitilis AVD2-14 by conjugal transfer. The double cross-over recombinant strain AV-C337 was selected and the fermentation products of this strain was analyzed. Results showed that it only produced two components of avermectins which were B_2a and B_1a respectively, and the useless "A" components completely disappeared. However, the yield of fermentation products of the recombinant strain obviously decreased.In order to avoid the decreasing of fermentation products caused by inserting long gene fragment, a mutation deleting five conservative amino acids was introduced into aveD by更多还原