【作者】 王洪滨；

【导师】 张利平；

【作者基本信息】 河北大学 ， 微生物学， 2004， 硕士

【摘要】 本论文首先介绍了阿维链霉菌生物合成的研究进展。由阿维链霉菌产生的阿维菌素是目前发现的最有效的杀螨虫、寄生虫和节肢昆虫的药剂之一。它包括8个组分，其中B1组分最为有效。阿维菌素是一种次级代谢产物生物合成复杂，现在已对每一步合成途径和其中重要的酶有所了解。随着分子生物学技术的发展，目前，人们已经完成了阿维链霉菌全基因组的测定，找到了30个与阿维链霉菌次级代谢有关的基因簇，掌握了整个阿维菌素合成的主要基因簇。这些使人们更加便于从分子角度对阿维菌素的生物合成进行调节，以获得高产高效的阿维链霉菌。另外许多新的选育技术也应用于阿维链霉菌菌种的选育。本实验室已应用我国的成熟的离子注入技术选育并获得高产阿维链霉菌。另外，随着宇航技术的成熟，越来越多的人认识到太空复杂的环境是地球上所无法比拟的，其产生的诱变效果也是地球上无法比拟的。因此应用宇航搭载技术筛选高产生产菌株吸引了越来越多人的关注。 在试验中发现铵离子对阿维菌素的生物合成有强烈的抑制作用，且铵离子可增加阿维菌素A组分的百分含量。因此获得对铵离子有较高耐受能力的菌株，则可增加阿维菌素的产量及提高B组分的比例。另外已经证实阿维菌素A组分C5位氧甲基由甲硫氨酸提供且甲硫氨酸可促进S-腺苷甲硫氨酸(SAM)的合成，故希望通过选择利用甲硫氨酸能力较强的菌株，使甲硫氨酸尽可能用于能量代谢，从而减少SAM前体的供给，以获得B组分含量较高的菌株。由磺胺药物的作用机制可知，对磺胺敏感的菌株转运一碳单位的能力也可能相对较弱，因此其体内SAM含量可能相对较低，这样的菌株所合成的阿维菌素中B组分的含量可能会有所提高。基于这三方面的考虑，本试验首先将经过离子注入诱变的高产菌株Z138经亚硝基胍诱变后分别做耐CsCl，耐甲硫氨酸和对磺胺敏感菌株的筛选，得到高产且效价稳定的5株菌株。它们是A22，D5，D6，77和73，效价分别为4974μg／mL，4214μg／mL，4930μg／mL，4410μg／mL，4369μg／mL，比出发菌株分别提高64.87％，28.48％，51.58％，16％，15％。 摘要二...曰吕..。vermj tz’jfs) HYI进行飞船搭载后，共分离纯化单菌落1237株，发酵测定618株菌，经过初筛和复筛，得到了24株高产且遗传性状稳定的菌株。这24株高产菌株的总效价都比出发菌株HYI提高24%以上，最高的提高了83%。同时，用薄层层析法和H尸LC法筛选Bla组分较高菌株，最终得到组分优化的高效菌株2株，其中菌株卜IY365的总效价为5924.67 pg/mL，它的Bla含量为3892“g/mL，占总效价的比例达到65.74%;HY516的Bla组分的含量为3076协g/mL，占总效价的59.9%。 在进行Bla组分较高菌株筛选的同时又对高产菌株HY365进行了发酵培养基的优化，发现玉米淀粉和花生饼粉对发酵结果影响极大:找到了适合H丫365的培养基组合7。另外还进行了前体物质对菌体发酵效价影响的试验，发现发酵时在培养基中加入一定浓度的前体物质丙酸钠，可以显著提高发酵产量，其中在培养基中加入2. smmol/L的前体物质丙酸钠，发酵产量可达到6424”g/mL。更多还原

【Abstract】 The avermectins from Streptomyces avermitilis are the most effective agents to kill helminthes, nematode and arthropod parasites at present. They are eight components, and the Bl component has the most effective antiparasitic activity. Avermectins are the secondary metabolites and its biosynthesis is very complex. Now the biosynthetic pathway of the avermectins and the most important enzymes have been found. With the development of molecular biology, complete genome sequence has been determined and 30 gene clusters related with avermectins biosynthesis have been got and the key gene clusters have been achieved. Because of these, the biosynthesis of avermectins can be regulated in the point of molecular so that the high-yield strains are obtained. What is more, many new technologies have also been used to breed the strains of Streptomyces avermitilis. In our lab the ion implanting technology has been used and the high-yield strains have been got. More and more people realized that the environment of space is so complex that the mutation result can not be imagined on the earth. Therefore the technology of screening the high-yield strains carried by spaceship has attracted more and more people.In the experiment NH4+ with the ability of inhibition the biosynthesis of avermectins can increase the percent of A components of avermectins. So the strains of anti- NH4+ can enhance the yield of avermectins and the ratio of B components of avermectins. It has been confirmed that methionine offers C5-O methylation to A components of avermectins and can help to synthesize S-adenosylmethionine(SAM). So the strains that can use methionine to metabolism are hoped to be get so that the strains with high percent of B components are got. According to the mechanism, strains sensitive to sulfanilamide are bad at transferring methyl, so there are lower of SAM in the strains and the B components may increase. Because of these reasons, after mutation by ion implanting strains Z138 was mutated by NTG combined with the ways of chlorocaeaium tolerance, methionine tolerance and being sensitive to sulfanilamiderespectively. 5 high-yield strains with genetic stability, namely A22, D5,D6,77 and 73, were obtained. Their titers were 4974?g/mL, 4214?g/mL, 4930 ?g/mL, 4410?g/mL, 4369 u g/mL, respectively. Compared with the original strain, the titers were improved 51.58%, 28.48%, 64.87%, 16%, 15% respectively.After mutation of ion implanting and NTG strain HY1 was carried by spaceship. Then 1237 mutant strains were isolated and purified.24 high-yield strains with genetic stability were got by two rounds screening. Their titers all increased over 24% and the highest one reached 83%. By the thin layer chromatograph(TLC) and high pressure and liquid chromatograph(HPLC), 2strains with high ratio of Bla components were obtained. The liter of strain HY365 reached to 5924.67 ?g/mL and Bla component was 3892 ?g/mL,65.74% of the tiler. And the Bla component of strain HY516 was 3076 u g/mL,59.9%ofthetiter.An optimizing medium composition 7 was found via orthogonal experimenl and the resull was confirmed that corn starch and peanut influenced the liter of fermentation. Experiment on the influence of precursor was made and the liter can increase evidently. When ihe concenlralion of the precursor reached to 2.5mmol/L, the tiler of avermeclins was 6424 u g/mL.更多还原