【摘要】：作為一種重要的平臺(tái)化合物,三羥基丙酸(3-HP)的應(yīng)用非常廣泛。目前三羥基丙酸主要是依靠化學(xué)合成的方法進(jìn)行生產(chǎn),這將對(duì)環(huán)境保護(hù)和可持續(xù)發(fā)展產(chǎn)生不利影響。藍(lán)細(xì)菌擁有可以直接固定CO2的能力,使其成為適合燃料以及化學(xué)品生產(chǎn)的自養(yǎng)型微生物“細(xì)胞工廠”。在本研究中,我們?cè)谒{(lán)細(xì)菌集胞藻Synechocystis sp.PCC 6803中構(gòu)建了三羥基丙酸的生物合成途徑,并通過(guò)以下方法對(duì)該生物合成系統(tǒng)進(jìn)行了優(yōu)化:1)使用不同啟動(dòng)子提高丙二酰輔酶A還原酶(MCR)基因表達(dá)量以及培養(yǎng)條件的優(yōu)化;2)過(guò)表達(dá)乙酰輔酶A羧化酶和生物素�；富蛱岣咔绑w物丙二酰輔酶A產(chǎn)量;3)過(guò)表達(dá)NAD(P)轉(zhuǎn)氫酶基因改善NADPH供應(yīng);4)通過(guò)敲除PHA以及乙酸合成競(jìng)爭(zhēng)通路使得碳源更多的流入3-HP合成通路。通過(guò)優(yōu)化,經(jīng)過(guò)6天的培養(yǎng)Synechocystis中3-HP產(chǎn)量可以達(dá)到837.18 mg L-1(348.8 mg/g細(xì)胞干重)。另外,過(guò)表達(dá)來(lái)自魚(yú)腥藻Anabaena sp.PCC7120和聚球藻Synechococcus sp.PCC 7942中核酮糖-1,5-二磷酸羧化酶/加氧酶基因并沒(méi)有增加3-HP產(chǎn)量,這表明CO2固定也許并不是影響3-HP的生物合成的主要因素。目前對(duì)于藍(lán)細(xì)菌細(xì)胞內(nèi)部3-HP生物合成對(duì)代謝的響應(yīng)機(jī)制的研究尚無(wú)報(bào)道。為此,在Synechocystis中成功構(gòu)建了3-HP代謝通路后,我們利用同位素標(biāo)記相對(duì)和絕對(duì)定量技術(shù)與液相二級(jí)質(zhì)譜聯(lián)用技術(shù)(iTRAQ-LC-MS/MS)對(duì)3-HP生產(chǎn)株與野生型中蛋白表達(dá)譜進(jìn)行了比較。經(jīng)過(guò)比對(duì),蛋白組分析共檢出2,264個(gè)獨(dú)立蛋白,占Synechocystis基因組預(yù)測(cè)蛋白總數(shù)的63%。在所有檢測(cè)到的蛋白中上調(diào)蛋白為204個(gè),下調(diào)蛋白為123個(gè)。我們還利用液相色譜與質(zhì)譜聯(lián)用技術(shù)(LC-MS)對(duì)細(xì)胞內(nèi)24種重要中心代謝物進(jìn)行了檢測(cè),綜合蛋白組學(xué)與代謝組學(xué)的數(shù)據(jù)可以看出Synechocystis中有關(guān)光合作用,氧化磷酸化作用,中心碳代謝過(guò)程的蛋白質(zhì)以及轉(zhuǎn)運(yùn)結(jié)合蛋白,蛋白翻譯過(guò)程,細(xì)胞調(diào)控過(guò)程的蛋白以及中心碳代謝過(guò)程中24種重要代謝物中有14種代謝物上調(diào)。綜合蛋白組和代謝組的數(shù)據(jù)我們發(fā)現(xiàn)向集胞藻中引入外源3-HP合成途徑后,由于大量生產(chǎn)3-HP,細(xì)胞需要通過(guò)代謝調(diào)控機(jī)制對(duì)系統(tǒng)內(nèi)所消耗的前體物以及能量、還原力進(jìn)行補(bǔ)充,并通過(guò)自我調(diào)節(jié)降低3-HP產(chǎn)生毒性的影響,以保證了細(xì)胞在正常生長(zhǎng)。我們還通過(guò)RT-qPCR分析和相關(guān)基因的過(guò)表達(dá)對(duì)蛋白組數(shù)據(jù)以及代謝調(diào)控機(jī)制進(jìn)行了驗(yàn)證,結(jié)果進(jìn)一步證明了蛋白組數(shù)據(jù)的可靠性以及機(jī)理分析的準(zhǔn)確性。通過(guò)對(duì)3-HP生產(chǎn)株與野生型的代謝和網(wǎng)絡(luò)分析比較使我們對(duì)Synechocystis sp.PCC 6803中3-HP的代謝響應(yīng)機(jī)制有了更深刻的認(rèn)識(shí)。
[Abstract]:As an important platform compound, 3-hydroxypropionic acid (3-HP) is widely used. At present, the production of trihydroxypropionic acid mainly depends on chemical synthesis, which will have adverse effects on environmental protection and sustainable development. Cyanobacteria have the ability to fix CO2 directly, making it suitable for fuel and chemical production. In this study, we constructed the biosynthetic pathway of trihydroxypropionic acid in Synechocystis sp. PCC 6803, and optimized the biosynthetic system by the following methods: 1) using different promoters to increase the expression of malonyl coenzyme A reductase (MCR) gene and the expression of MCR gene. Optimization of culture conditions; 2) Overexpression of acetyl-CoA carboxylase and biotin acylase genes increased the production of the precursor malonyl-CoA; 3) Overexpression of NAD (P) transhydrogenase gene improved the supply of NADPH; 4) Knocking out the competition pathway of PHA and acetic acid synthesis increased the flow of carbon into the 3-HP synthesis pathway. In addition, overexpression of ribose-1,5-diphosphate carboxylase/oxygenase gene from Anabaena sp. PCC7120 and Synechococcus sp. PCC 7942 did not increase 3-HP production, suggesting that CO2 fixation may not be the main factor affecting the biosynthesis of 3-HP. Essential factors. Up to now, no studies have been reported on the mechanism of 3-HP biosynthesis in cyanobacterial cells in response to metabolism. A total of 2,264 independent proteins were detected by proteomic analysis, accounting for 63% of the total predicted proteins in the Synechocystis genome. Of all the proteins detected, 204 were up-regulated and 123 were down-regulated. Liquid chromatography-mass spectrometry (LC-MS) was also used to identify 24 important intracellular proteins. Cardiac metabolites were detected. Proteomics and metabonomics data showed that 24 important metabolites in Synechocystis, including photosynthesis, oxidative phosphorylation, central carbon metabolism proteins and transport-binding proteins, protein translation, cell-regulated proteins, and central carbon metabolism, were involved. 14 metabolites are up-regulated. Data from proteomics and metabolomics show that after introducing exogenous 3-HP biosynthesis pathway into Syncystis spp., cells need to replenish the precursors and energy consumed by the system through metabolic regulation mechanism, and reduce the toxicity of 3-HP by self-regulation. We also validated the proteomic data and metabolic regulation mechanism by RT-q PCR analysis and overexpression of related genes. The results further proved the reliability of proteomic data and the accuracy of mechanism analysis. We have a deeper understanding of the metabolic response mechanism of 3-HP in Synechocystis sp.PCC 6803.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q946;TQ225.4

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