多基因同时调控提高β-胡萝卜素产量
发布时间：2019-01-08 18:00
【摘要】：代谢合成途径优化的关键在于途径中多个基因表达的调控,尤其是如何对多基因同时调控。本研究针对β-胡萝卜素的生产,分别对β-胡萝卜素合成途径的上游和下游途径进行了多基因同时调控。为了β-胡萝卜素上游合成途径的调控,将来源于E.faecalis和S.Pneumoniae的MVA途径的5个基因mvaE、mvaS、mvaK1、mvaD、mvaK2引入实验室之前构建的β-胡萝卜素生产菌CAR005中。并使用RBS文库对这五个基因进行调控,配合Golden Gate组装方法,可以通过一次Golden Gate酶切连接反应得到带有不同调控模式的通路基因的质粒文库。将这些通过特殊设计得到的质粒文库转化到CAR005中。通过β-胡萝卜素的颜色初筛,分光光度法测定产量的复筛,我们从中得到一系列产量提高幅度高低不同的有代表性的菌株,其中产量最高的菌株其β-胡萝卜素产量比对照提高129.2%。选取部分菌株测序,证明了质粒文库确实包含着mvaE、mvaS、mvaK1、mvaD、mvaK2的不同表达类型。用软件计算MVA基因对应RBS的理论强度,并对MVA途径5个基因RBS强度组成情况进行分析。类似地,对于β-胡萝卜素下游合成途径的调控,通过Golden Gate DNA组装方法构建质粒,使用不同强度的启动子对来源于P.agglomerans的β-胡萝卜素合成途径的下游基因crtE、crtY、crtI、crtB进行了同时调控,从而优化代谢通路。通过颜色筛选,β-胡萝卜素产量定量测定,得到一系列产量提高幅度高低不同的菌株,产量最高的菌株比对照提高65.2%。通过测序,得到菌株crtE、crtY、crtI、crtB基因前启动子组合情况,并对其分析。本研究验证了通过RBS文库或是不同强度启动子,搭配Golden Gate质粒构建方法建库对代谢通路多个基因同时调控以提高目的产物产量的可行性,同时也进一步证明了多基因同时调控的必要性。
[Abstract]:The key to the optimization of metabolic biosynthesis pathway lies in the regulation of multiple genes expression, especially how to regulate the multiple genes at the same time. In order to produce 尾 -carotene, the upstream and downstream pathways of 尾 -carotene biosynthesis were regulated simultaneously. In order to regulate the upstream biosynthesis pathway of 尾 -carotene, five mvaE,mvaS,mvaK1,mvaD,mvaK2 genes derived from the MVA pathway of E.faecalis and S.Pneumoniae were introduced into the 尾 -carotene producing strain CAR005. The five genes were regulated by RBS library, and the plasmid library with different regulatory patterns could be obtained by a Golden Gate ligation reaction with the method of Golden Gate assembly. These plasmids obtained by special design were transformed into CAR005. Through the primary screening of 尾 -carotene and the double screening of the yield determined by spectrophotometry, we obtained a series of representative strains with different yield increases. The 尾-carotene production of the highest yield strain was 129.2% higher than that of the control strain. Some strains were sequenced and it was proved that the plasmid library did contain different expression types of mvaE,mvaS,mvaK1,mvaD,mvaK2. The theoretical strength of MVA gene corresponding to RBS was calculated by software, and the composition of RBS intensity of five genes of MVA pathway was analyzed. Similarly, for the regulation of the downstream synthetic pathway of 尾 -carotene, the plasmid was constructed by Golden Gate DNA assembly method, and the downstream gene crtE,crtY,crtI, derived from the 尾 -carotene synthesis pathway was constructed using different intensities of promoters. CrtB was simultaneously regulated to optimize the metabolic pathway. By color screening and quantitative determination of 尾 -carotene yield, a series of strains with different yield increasing range were obtained, and the highest yield strains increased 65.2% compared with the control. By sequencing, the prepromoter combination of crtE,crtY,crtI,crtB gene was obtained and analyzed. This study demonstrated the feasibility of simultaneous regulation of multiple genes in metabolic pathway by using RBS library or promoter with different intensities, using Golden Gate plasmid to construct the library to increase the yield of the target product. At the same time, it also proved the necessity of simultaneous regulation of polygenes.
【学位授予单位】：大连工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS201.3
[Abstract]:The key to the optimization of metabolic biosynthesis pathway lies in the regulation of multiple genes expression, especially how to regulate the multiple genes at the same time. In order to produce 尾 -carotene, the upstream and downstream pathways of 尾 -carotene biosynthesis were regulated simultaneously. In order to regulate the upstream biosynthesis pathway of 尾 -carotene, five mvaE,mvaS,mvaK1,mvaD,mvaK2 genes derived from the MVA pathway of E.faecalis and S.Pneumoniae were introduced into the 尾 -carotene producing strain CAR005. The five genes were regulated by RBS library, and the plasmid library with different regulatory patterns could be obtained by a Golden Gate ligation reaction with the method of Golden Gate assembly. These plasmids obtained by special design were transformed into CAR005. Through the primary screening of 尾 -carotene and the double screening of the yield determined by spectrophotometry, we obtained a series of representative strains with different yield increases. The 尾-carotene production of the highest yield strain was 129.2% higher than that of the control strain. Some strains were sequenced and it was proved that the plasmid library did contain different expression types of mvaE,mvaS,mvaK1,mvaD,mvaK2. The theoretical strength of MVA gene corresponding to RBS was calculated by software, and the composition of RBS intensity of five genes of MVA pathway was analyzed. Similarly, for the regulation of the downstream synthetic pathway of 尾 -carotene, the plasmid was constructed by Golden Gate DNA assembly method, and the downstream gene crtE,crtY,crtI, derived from the 尾 -carotene synthesis pathway was constructed using different intensities of promoters. CrtB was simultaneously regulated to optimize the metabolic pathway. By color screening and quantitative determination of 尾 -carotene yield, a series of strains with different yield increasing range were obtained, and the highest yield strains increased 65.2% compared with the control. By sequencing, the prepromoter combination of crtE,crtY,crtI,crtB gene was obtained and analyzed. This study demonstrated the feasibility of simultaneous regulation of multiple genes in metabolic pathway by using RBS library or promoter with different intensities, using Golden Gate plasmid to construct the library to increase the yield of the target product. At the same time, it also proved the necessity of simultaneous regulation of polygenes.
【学位授予单位】：大连工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS201.3
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