【作者】 郑子静；

【导师】 郭美锦；

【作者基本信息】 华东理工大学 ， 生物化工， 2011， 硕士

【摘要】 土霉素(Oxytetracycline, OTC)是在动物和水产品饲料中应用广泛的重要抗生素,基因调控策略是提高抗生素生物合成的有效方法之一。为此,本论文以工业土霉素产生菌-龟裂链霉菌(Streptomyces rimosus)为研究对象,分别通过初级代谢途径关键酶改造和增强自身抗性基因剂量方法,研究提高土霉素(OTC)生物合成的策略。基于初级代谢途径的改变可能会触发次级代谢水平以及细胞生理变化,且结合本实验室以前通过龟裂链霉菌模式菌株M4018中葡萄糖-6-磷酸脱氢酶(Glucose-6-phosphate dehydrogenase, G6PDH)失活来增加土霉素产量的研究,本论文研究了G6PDH编码基因zwfl失活对龟裂链霉菌工业菌(SRI)的影响,得到如下结果：(1)构建得到一株G6PDH失活的土霉素高产菌株S-z8,其OTC产量较原始出发菌株SRI提高36.2%；(2)将SRI与S-z8发酵生理代谢比较,发现细胞生长到第6天时,SRI的生物量较S-z8高90%,而两者的耗糖速率却没有显著差异。另外,本论文从次级代谢角度出发,研究SRI自身抗性基因otrA与otrB拷贝数增加对土霉素生物合成影响。得到如下结果：(1)通过位点特异性整合将otrA和otrB分别整合到SRI染色体上,构建筛选得到一株OTC高产突变株S-B80；(2)SRI与S-B80两株菌株土霉素对于底物的得率YP/s(mg/g), S-B80比SRI高81.83%；生物量对于底物的得率Yx/s(mg/g) S-B80却较SRI降低了38.58%；(3)摇瓶发酵表明,S-B80胞内OTC浓度较SRI降低13.8%,同时S-B80胞内外OTC浓度总和比SRI高14.2%,说明otrB拷贝数增加可提高细胞外排土霉素至胞外的能力,也具有提高土霉素生物合成的作用。因此,通过分别对初级代谢中zwfl基因的失活和OTC自身抗性基因otrB拷贝数增加,都能有效提高土霉素的生物合成,为实际工业生产提供了重要基础。更多还原

【Abstract】 The aromatic polyketide antibiotic oxytetracycline (OTC) produced by Streptomyces rimosus is one of important secondary polyketide metabolites.Currently, regulatory gene manipulation has been developed one of effective ways to improve antibiotic biosynthesis. Taken industrial Streptomyces rimosus(SRI) strain as an example, in this thesis the investigation of gene disruption of a key enzyme in primary metabolic pathways and enhancement of the cell’s self-resistant genes were carried out to improve the OTC productivity.Based on the facts that perturbation in primary metabolic pathways can affect both the secondary metabolism and physiological characterization, and that zwf2 gene knock-out in a type strain M4018 enhanced OTC production in our previous work, zwfl encoding Glucose-6-phosphate dehydrogenase(G6PDH) was disrupted to explore its effects on oxytetracycline biosynhtesis in an industrail OTC producer SRI. The results were obtained as follows:(1) A mutant with zwf1 disruption, designated S-z8, was genetically constructed and successfully screened, The maximum oxytetracycline level in S-z8 was 36.2%higher than that in parent strain SRI.(2) Experimental results showed that the biomass of SRI was 90%more than S-z8 on the sixth day when cultured in the shaking flask, however, there were no significant differences in substrate consumption rate between mutant and parent strain.In addition, to improve OTC production, extra copy of two self-resistant genes otrA and otrB was introduced into SRI OTC, respectively, the results were as follows:(1) otrA or otrB gene was introduced into SRI genome by site-specific integration at attB site and a mutant with high-yield OTC performance was screened, and designated S-B80;(2) OTC Yield on substrate (YP/S,mg/g) of S-B80 was 81.83%higher than that of SRI whereas Yx/s(mg/g) of the mutant was 38.58%lower than SRI;(3) Compared with SRI, the intracellular OTC contents in SRI-B80 was 13.8% lower, however, total OTC produced by SRI-B80 was 14.2%higher, indicating that otrB could exflux intracelluler OTC into ouside the cell so that it could elevated OTC production by SRI.更多还原