【作者】 陈川；

【导师】 张利平；

【作者基本信息】 河北大学 ， 微生物学， 2002， 硕士

【摘要】 本文首先介绍了阿维菌素的生物合成与研究概况。阿维菌素是迄今已发现最有效的杀昆虫剂、杀螨虫剂和杀寄生虫剂之一。阿维菌素为一种典型的次级代谢产物，生物合成途径复杂，现在基本上对每一步合成途径的基因及其所编码的酶都有所了解。这使得人们可利用基因工程技术来构建工程菌，增加有效组分的产出和生产新型的阿维菌素。除了用生物方法，人们还通过化学修饰来产生抗虫活性更高的阿维菌素衍生物。本论文主要为诱变筛选阿维菌素高产菌株，并对筛选到的高产菌株进行了培养条件与培养基成分的优化，为工业化生产打下基础。 本研究探讨了阿维链霉菌严重的自然分化现象，在平板分离中存在4种不同的菌落形态分化菌株，其中灰色孢子菌落占大多数，发酵效价最高；只有气生菌丝的白色菌株和只有基内菌丝的光秃型菌落占少数，还有一些灰色菌落中有白点，这些菌落发酵效价较低。这种有白点的菌落形态与灰色菌落类似，可能为放线菌中形成的异核体。白色菌株和光秃型菌株经传代培养比较稳定，灰色有白点的菌落在传代过程中不断分化出灰色、白色、光秃型菌落，直接造成遗传的不稳定性，使生产效价不稳定，为生产的大害。 在育种研究中应用了离子注入、亚硝基胍和紫外线诱变方法，并对这几种诱变方法进行了比较，发现离子注入的诱变效果最好。离子注入后阿维链霉菌的菌落形态发生了很大的变化，其中灰色，边缘整齐，中间突出有开裂的菌落和为灰色，边缘波浪状，中间突出有放射状开裂的菌株产量较高。通过两轮诱变筛选出最终得到了8株发酵效价提高并且遗传性能稳定的突变株。分别是A208、C56、B260、Z138、N10、N301、S3、E3，总效价比出发菌株提高了16.9％-63％。突变株Z138总效价最高达3900μg／ml。其中突变株N10的B_1a的比例提高了9.6％。 以筛选到的突变菌株Z138为实验菌株，通过摇瓶确定了发酵的工艺条件。发酵时间9-10天开始迅速合成阿维菌素；温度28℃最好；接种量12％使阿维菌素的产量较高；转速210转／分较为适合产生阿维菌素的通气量；选用30小时左右的种 MX龄可使阿维菌素的产量提高。！t离子抑制阿维菌素的合成：在一定浓度下KZHPO4和MgSO。对阿维菌素的合成没有影DDI(u。 培养基的优化经历了两个步骤，首先迎过单因素实验了解了培养基成分对D：I给菌素发酵的影响，然后通过正交分析对发酵培养基进行了优化，认为玉米淀粉，处上讲粉与玉米浆对阿维菌素的合成影响较大，并仰定了”g／1的玉米淀粉、Zg／1玉米浆，10 g／l黄豆饼粉的发酵培养基。发酵培养基优化后使突变株 ZI3 8的阿细菌素产量达到了4200ug／ml左右。更多还原

【Abstract】 The avermetins are a group of closely related macrocylic lactones with exceedingly high activity against helminths and anthropods. This paper review the biosyntheticpathway of the avermectins and the organization of the biosynthetic gene cluster which many groups have analysed and cloned. According to the cloned gene cluster, people can use genetical technology to obtain the genetical strains which can produce more potent and non-toxical avermectins and its derivatives. An extensive program of chemical modificaton can also be carried out to discover more active avermectins. Avermectin has been into industrial produce as one of wide-scope pesticide.Four types of colony ,powdery gray, gray with white blot ,white and bald were isolated from Streptomyces avermitilis among them only the powdery gray one produced the highest avermectin. The Gray with white blot one only produced 70%-80% less than the powdery gray. And the others produced none.The effects of various mutagenic agents on the productivity of avermectin by Slreplomyces avermilitis were evaluated in this work. Three different mutagenic breeding methods were developed, such as NTG, UV, Ion implatation. Ion implantation were most efficient for positive mutation of S. avermilitis. The distribution fractions of mutants with higher avermectin titer than the original had correlation with implantation dose. After ion implantation mutation there were many types of morphology of colony , we stdudied the relationship between the colony shape and the ability of yielding , found that gray and protuberant colony had the highest titer.After two rounds of cyclic screening, we obtained eight mutant strains which produced 16.9-63.2% more avermectin than that of original strain. And they were all genetic stability. Its avermectin titer attained 3900ug/ml in shaking flask. B1a ratio of Strain N10 attained 29.9%.Several factors affecting avermectin production were studied. The highest avermectin yielding were obtained when the fermentation conditions were as follows: temperature 28@, inoculation 12%, rotation 210 r.p.m, seed age of 30h. It was found that the ammonia ion could distinctly inhibit biosynthesis of avermectin and the mechanism of this phenomenon was investigated. K2HPO4 and MgSO4 did not inhibit orincrease the avermectin yielding.Comprehensive studies were carryied out in composing the fermentation medium. An optimizing medium was obtained via orthogonal experiment. The optimized concentration : cornsteep 2g/l, corn starch 90g/l and soybean cakepowder 10g/l.更多还原