【摘要】：代謝合成途徑優(yōu)化的關(guān)鍵在于途徑中多個基因表達的調(diào)控,尤其是如何對多基因同時調(diào)控。本研究針對β-胡蘿卜素的生產(chǎn),分別對β-胡蘿卜素合成途徑的上游和下游途徑進行了多基因同時調(diào)控。為了β-胡蘿卜素上游合成途徑的調(diào)控,將來源于E.faecalis和S.Pneumoniae的MVA途徑的5個基因mvaE、mvaS、mvaK1、mvaD、mvaK2引入實驗室之前構(gòu)建的β-胡蘿卜素生產(chǎn)菌CAR005中。并使用RBS文庫對這五個基因進行調(diào)控,配合Golden Gate組裝方法,可以通過一次Golden Gate酶切連接反應(yīng)得到帶有不同調(diào)控模式的通路基因的質(zhì)粒文庫。將這些通過特殊設(shè)計得到的質(zhì)粒文庫轉(zhuǎn)化到CAR005中。通過β-胡蘿卜素的顏色初篩,分光光度法測定產(chǎn)量的復(fù)篩,我們從中得到一系列產(chǎn)量提高幅度高低不同的有代表性的菌株,其中產(chǎn)量最高的菌株其β-胡蘿卜素產(chǎn)量比對照提高129.2%。選取部分菌株測序,證明了質(zhì)粒文庫確實包含著mvaE、mvaS、mvaK1、mvaD、mvaK2的不同表達類型。用軟件計算MVA基因?qū)?yīng)RBS的理論強度,并對MVA途徑5個基因RBS強度組成情況進行分析。類似地,對于β-胡蘿卜素下游合成途徑的調(diào)控,通過Golden Gate DNA組裝方法構(gòu)建質(zhì)粒,使用不同強度的啟動子對來源于P.agglomerans的β-胡蘿卜素合成途徑的下游基因crtE、crtY、crtI、crtB進行了同時調(diào)控,從而優(yōu)化代謝通路。通過顏色篩選,β-胡蘿卜素產(chǎn)量定量測定,得到一系列產(chǎn)量提高幅度高低不同的菌株,產(chǎn)量最高的菌株比對照提高65.2%。通過測序,得到菌株crtE、crtY、crtI、crtB基因前啟動子組合情況,并對其分析。本研究驗證了通過RBS文庫或是不同強度啟動子,搭配Golden Gate質(zhì)粒構(gòu)建方法建庫對代謝通路多個基因同時調(diào)控以提高目的產(chǎn)物產(chǎn)量的可行性,同時也進一步證明了多基因同時調(diào)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.
【學(xué)位授予單位】：大連工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TS201.3

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