【摘要】：隨著世界經(jīng)濟(jì)的不斷發(fā)展,能源短缺與環(huán)境污染問題逐漸顯現(xiàn),如何開發(fā)可持續(xù)、清潔能源并緩解環(huán)境污染至為重要。藍(lán)細(xì)菌是光合自養(yǎng)微生物,通過光合作用固定二氧化碳合成有機(jī)物并釋放氧氣,藍(lán)細(xì)菌合成生物燃料不僅可以緩解能源短缺問題,還可利用化石燃料燃燒釋放的二氧化碳進(jìn)而減輕溫室效應(yīng)。本研究根據(jù)藍(lán)細(xì)菌代謝途徑特點(diǎn),通過基因工程手段探究合成生物乙醇的優(yōu)化策略,研究內(nèi)容和結(jié)果如下:(1)以集胞藻PCC 6803的單拷貝工程藻株Syn-ZG25(基因組slr0168位點(diǎn)表達(dá)了運(yùn)動發(fā)酵單胞菌的丙酮酸脫羧酶基因PDCzm和集胞藻PCC 6803的乙醇脫氫酶基因slr1192)為基礎(chǔ),在基因組phaAB位點(diǎn)過表達(dá)一份PDCzm得到工程藻株Syn-YQ4,其乙醇產(chǎn)量與雙拷貝藻株Syn-HZ24(slr0168和pha AB兩個位點(diǎn)同時表達(dá)PDCzm和slr1192)相當(dāng),通過酶活和Western Blot結(jié)果分析PDCzm的表達(dá)量和活性為乙醇合成途徑的限制性因素。(2)聚球藻PCC 7002中構(gòu)建的乙醇合成藻株通過Western Blot顯示slr1192表達(dá)量明顯高于PDCzm,于是分別在野生型中通過強(qiáng)啟動子控制slr1192和產(chǎn)乙醇藻株中過表達(dá)一份slr1192,其中強(qiáng)啟動子控制方案無效,過表達(dá)slr1192后酶活和表達(dá)量均明顯提高。(3)以目前文獻(xiàn)報道倍增時間最短的藍(lán)細(xì)菌聚球藻UTEX 2973為基礎(chǔ),在其中性位點(diǎn)NS1分別正向和反向表達(dá)一份PDCzm和slr1192,成功構(gòu)建產(chǎn)乙醇藻株Syn-YQ16(正向插入)和Syn-YQ17(反向插入),其中Syn-YQ16較Syn-YQ17乙醇產(chǎn)量略高,但乙醇合成速率較PCC 6803和PCC 7002單拷貝藻株偏低。
[Abstract]:With the development of the world economy, the problems of energy shortage and environmental pollution appear gradually. How to develop sustainable, clean energy and mitigate environmental pollution is very important. Cyanobacterium is a photosynthetic autotrophic microorganism, which immobilizes carbon dioxide to synthesize organic matter and release oxygen through photosynthesis, and synthesizing biofuel by cyanobacteria can not only alleviate the problem of energy shortage. The burning of fossil fuels can also be used to reduce carbon dioxide emissions in Greenhouse Effect. According to the characteristics of cyanobacteria metabolic pathway, the optimization strategy of biosynthesis of bioethanol was explored by genetic engineering. The main contents and results were as follows: (1) based on the single copy engineering algae strain Syn-ZG25 of PCC 6803, which expressed pyruvate decarboxylase gene (PDCzm) at genomic slr0168 site and ethanol dehydrogenase gene slr1192 (slr1192 gene of PCC 6803). An engineering algae strain Syn-YQ4, was obtained by overexpression of a PDCzm at the genomic phaAB site. The ethanol production of Syn-YQ4, was comparable to that of Syn-HZ24 (slr0168 and pha AB expressed PDCzm and slr1192 at both slr0168 and pha AB loci). The expression and activity of PDCzm were analyzed by enzyme activity and Western Blot results. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. (2) the expression of slr1192 in PCC 7002 was significantly higher than that in PDCzm,. So one slr1192, was expressed in wild type by strong promoter control slr1192 and ethanol producing algae strain respectively, and the strong promoter control scheme was not effective. (3) on the basis of UTEX 2973, which has the shortest doubling time reported in the literature, a PDCzm and slr1192, were expressed in the neutral NS1 of Chlorella cyanobacterium UTEX 2973, respectively. Syn-YQ16 (positive insertion) and Syn-YQ17 (reverse insertion) were successfully constructed. The ethanol production of Syn-YQ16 was slightly higher than that of Syn-YQ17, but the ethanol synthesis rate was lower than that of PCC 6803 and PCC 7002 single copy algae.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ223.122;TQ920.6

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本文編號：2429440