本文選題：生物燃料 + 藍藻��； 參考：《福建農(nóng)林大學》2017年碩士論文

【摘要】：世界人口增長和工業(yè)經(jīng)濟的發(fā)展對能源的需求越來越大,生物燃料作為一種可替代傳統(tǒng)燃料的環(huán)境友好型可再生能源受到了人們的關注。目前美國等發(fā)達國家和地區(qū)對生物燃料的重視程度和研究成績都遠領先于中國,主要的研究方向是以光合微生物為平臺生產(chǎn)人們需要的生物燃料和高價值產(chǎn)品。藍藻是作為第三代生物燃料的重要原料,利用太陽能固定CO2轉(zhuǎn)化成各種形式的生物能,極具應用前景。而萜類物質(zhì)家族龐大,能作為傳統(tǒng)燃料的替代品,還是應用廣泛的高價值化合物。因此,如何以藍藻為代謝平臺生產(chǎn)萜類物質(zhì)是這一領域的研究重點,然而現(xiàn)階段國內(nèi)關于這部分的研究仍較空白。本實驗以藍藻為實驗研究對象,在了解其MEP代謝路徑的基礎上,通過代謝工程調(diào)控方法,在藍藻 Cyanobacterium Synechococcus elongatus PCC7942表達高效的外源關鍵萜類合成酶基因(檸檬烯合成基因和法呢烯合成酶基因),在藍藻Synechocystissp.PCC 6803中驗證鯊烯環(huán)化酶基因敲除后的產(chǎn)物累計,以此探索增強或阻礙關鍵酶的表達對藍藻細胞萜烯產(chǎn)物累積和細胞生長的影響。目標產(chǎn)物分別包括單萜—檸檬烯,倍半萜—法尼烯和三萜-鯊烯。主要研究結果如下:1.通過構建含有檸檬烯合成酶基因LS的質(zhì)粒,并成功表達LS基因,我們證明了檸檬烯合成酶基因LS是檸檬烯的合成的關鍵,Cyanobacterium Synechococcus elongatus PCC7942 具備生產(chǎn)檸檬烯的能力。且不同的啟動子對相同基因的表達有影響,同時具有Ptrc啟動子和核糖體結合位點(RBS)的菌株產(chǎn)量最高,日平均產(chǎn)率達到53.0μg/L/OD/day,因此要提高產(chǎn)量需要篩選出高效的啟動子。2.通過構建含有法呢烯合成酶FS基因的質(zhì)粒,并成功表達FS基因通,我們證明了法呢烯合成酶基因FS是法尼烯合成的關鍵,且藍藻細胞PCC7942同樣具備法呢烯合成路徑中必要的原料和酶。3.驗證了鯊烯下游鯊烯藿烯環(huán)化酶基因Shc的敲除的確能積累鯊烯,藍藻突變株Synechocystissp.PCC 6803△Shc的鯊烯產(chǎn)率可達到2.48mg/L/OD,而在突變株的基礎上通過代謝工程進一步提高鯊烯產(chǎn)量,鯊烯合成基因或許是重要突破口。4.本實驗成功驗證了藍藻能產(chǎn)生單萜,倍半萜和三萜,藍藻作為原核光合自養(yǎng)型微生物具有眾多優(yōu)勢,完全能通過基因改造來實現(xiàn)萜烯的生產(chǎn)并且達到比較客觀的產(chǎn)量,為實現(xiàn)藍藻生物燃料的工業(yè)化生產(chǎn)提供了可能性。
[Abstract]:With the increase of world population and the development of industrial economy, more and more people pay attention to biofuel as a kind of environment-friendly renewable energy which can replace traditional fuel. At present, the United States and other developed countries and regions are far ahead of China in the importance and research achievement of biofuel. The main research direction is to produce biofuel and high-value products that people need on the platform of photosynthetic microbes. Cyanobacteria are important feedstock for the third generation biofuel, which can be converted into various forms of bioenergy by solar fixed CO2. The terpenoid family is so large that it can be used as a substitute for traditional fuels and as a high-value compound in a wide range of applications. Therefore, how to use cyanobacteria as metabolic platform to produce terpenoids is the focus of research in this field, but at present, the research on this part of the domestic is still relatively blank. In this study, cyanobacteria were selected as the experimental objects. On the basis of understanding their MEP metabolic pathway, the metabolic engineering regulation method was adopted. High efficient exogenous key terpene synthase genes (limonene synthase gene and farnene synthase gene) were expressed in cyanobacteria Cyanobacterium Synechococcus elongatus PCC7942. The accumulated products of squalene cyclase gene knockout were verified in cyanobacteria Synechocystissp.PCC 6803. To explore the effect of enhancing or blocking the expression of key enzymes on the accumulation of terpene products and cell growth in cyanobacteria. The target products include monoterpene-limonene, sesquiterpene-farnesene and triterpene-squalene respectively. The main results are as follows: 1. By constructing the plasmid containing LS gene and expressing LS gene successfully, we proved that LS gene is the key factor in limonene synthesis. Cyanobacterium Synechococcus elongatus PCC7942 has the ability to produce limonene. Different promoters affected the expression of the same gene, and the strains with Ptrc promoter and ribosomal binding site (RBS) had the highest yield, and the average daily yield was 53.0 渭 g / L / O ~ (-1) / day. therefore, to improve the yield, we should screen out highly efficient promoter. 2. By constructing a plasmid containing fellene synthase FS gene and expressing FS gene successfully, we have proved that fathene synthase gene FS is the key to farnesene synthesis. In addition, cyanobacteria cell PCC7942 also possesses the necessary raw materials and enzymes in the biosynthesis pathway of farnesene. It was proved that the knockout of squalene cyclase gene Shc in the downstream of squalene actually accumulated squalene. The squalene yield of the cyanobacteria mutant Synechocystissp.PCC 6803 Shc could reach 2.48 mg / L / O, and the squalene yield could be further increased by metabolic engineering on the basis of the mutant. Squalene synthesis gene may be an important breakthrough. 4. This experiment proved that cyanobacteria can produce monoterpenes, sesquiterpenes and triterpenes. Cyanobacteria have many advantages as prokaryotic photosynthetic autotrophic microorganisms. It provides the possibility for the industrial production of cyanobacteria biofuel.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q946

【參考文獻】

相關期刊論文 前10條

1 倪俊;陶飛;許平;;光合生產(chǎn)植物天然產(chǎn)物的代謝工程藍藻研究現(xiàn)狀[J];生物產(chǎn)業(yè)技術;2017年01期

2 王凌健;方欣;楊長青;李建戌;陳曉亞;;植物萜類次生代謝及其調(diào)控[J];中國科學:生命科學;2013年12期

3 姚美玲;侯和勝;;藻類光捕獲復合體LHC蛋白結構與功能的進化[J];天津農(nóng)業(yè)科學;2013年02期

4 ;藍藻生物燃料研究獲進展[J];河南化工;2012年17期

5 岳躍沖;范燕萍;;植物萜類合成酶及其代謝調(diào)控的研究進展[J];園藝學報;2011年02期

6 占愛瑤;由香玲;詹亞光;;植物萜類化合物的生物合成及應用[J];生物技術通訊;2010年01期

7 劉潤生;;美國先進生物燃料技術政策與態(tài)勢分析[J];中國生物工程雜志;2010年01期

8 鄧勇;陳方;王春明;陳云偉;房俊民;;美國生物質(zhì)資源研究規(guī)劃與舉措分析及啟示[J];中國生物工程雜志;2010年01期

9 虞功亮;樊慶春;余博識;郭嘉;王存文;李仁輝;;利用滇池水華藍藻提取生物燃料的方法比較[J];化學與生物工程;2009年04期

10 韓軍麗;李振秋;劉本葉;王紅;李國鳳;葉和春;;植物萜類代謝工程[J];生物工程學報;2007年04期

，

本文編號：1901934