本文關(guān)鍵詞： 2-羥基異丁酸 大腸桿菌 嗜水氣單胞菌 變位酶 發(fā)酵　出處：《北京化工大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：2-羥基異丁酸(2-Hydroxyisobutyric acid,簡(jiǎn)寫(xiě)2-HIBA)是一種有多用途的重要的化工產(chǎn)品,它可參與多種有機(jī)合成反應(yīng)生成一系列化合物,以它為平臺(tái)可以衍生出許多重要的其它化工產(chǎn)品,在許多領(lǐng)域有潛在的應(yīng)用前景。由于其結(jié)構(gòu)特殊,目前為發(fā)現(xiàn)其天然的微生物法合成途徑�；瘜W(xué)方法生產(chǎn)具有成本高、生成條件苛刻、污染大等缺點(diǎn),隨著合成生物學(xué)、分子生物學(xué)和代謝工程學(xué)等迅速發(fā)展,利用生物轉(zhuǎn)化的方法生產(chǎn)2-羥基異丁酸成為可能。本論文就微生物法生產(chǎn)2-羥基異丁酸進(jìn)行了初步的探索研究。以重組大腸桿菌為宿主,構(gòu)建了bktB、hbd、icmA、icmB和lesB等5個(gè)基因的表達(dá)載體pEnBHTI,以葡萄糖降解產(chǎn)生的乙酰CoA為前體,經(jīng)過(guò)4步代謝途徑,以期能夠合成2-羥基異丁酸。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),含有pEnBHTI的大腸桿菌能夠以葡萄糖為碳源獲得3-羥基丁酸,未檢測(cè)到明顯的2-羥基異丁酸積累,提示bktB、hbd和tesB成功表達(dá),但是icmAB表達(dá)量可能較低,或是tesB對(duì)2-羥基異丁酰輔酶A的催化活性較低,導(dǎo)致以重組大腸桿菌為宿主未能獲得產(chǎn)物。在此基礎(chǔ)上,以嗜水氣單胞菌為宿主,利用其脂肪酸氧化過(guò)程中產(chǎn)生的羥基丁酰輔酶A為前體,通過(guò)構(gòu)建icmA和icmB的表達(dá)載體pMCSV12,在嗜水氣單胞菌單胞菌中構(gòu)建2-羥基異丁酸的生物合成途徑。對(duì)重組菌株進(jìn)行搖瓶培養(yǎng),發(fā)現(xiàn)其能夠合成微量的2-羥基異丁酸；進(jìn)而采用不同的脂肪酸碳源、兩步法發(fā)酵等對(duì)培養(yǎng)條件進(jìn)行優(yōu)化,最終獲得了46 mg/L的產(chǎn)量。本論文為微生物法合成2-羥基異丁酸提供了初步的研究基礎(chǔ)。
[Abstract]:2-hydroxyisobutyric acid2-Hydroxyisobutyric acid2-Hydroxyisobutyric acid2-Hydroxyisobutyric acid (2-H@@. It can participate in a variety of organic synthesis reactions to form a series of compounds, it can be used as a platform to derive many important other chemical products, and has potential applications in many fields, because of its special structure. At present, in order to find its natural way of microbiological synthesis, chemical production has many disadvantages, such as high cost, harsh conditions and pollution. With the rapid development of synthetic biology, molecular biology and metabolic engineering, etc. It is possible to produce 2-hydroxyisobutyric acid by biotransformation. In this paper, the production of 2-hydroxyisobutyric acid by microbial method was studied. The recombinant Escherichia coli was used as host to construct bktB. The expression vector pEnBHTI of 5 genes, including hbddcicmAgnicmB and lesB, was used as precursor of acetyl CoA produced by glucose degradation, and was metabolized by four steps. In order to synthesize 2-hydroxyisobutyric acid, the results showed that 3-hydroxybutyric acid could be obtained by using glucose as carbon source in Escherichia coli containing pEnBHTI, but no obvious accumulation of 2-hydroxyisobutyric acid was detected. These results suggested that bktBnhbd and tesB were successfully expressed, but the expression of icmAB might be low, or the catalytic activity of tesB to 2-hydroxyisobutyryl coenzyme A was lower. On the basis of this, Aeromonas hydrophila was used as host and hydroxy butyryl coenzyme A produced during fatty acid oxidation was used as precursor. By constructing the expression vector pMCSV12 of icmA and icmB, the biosynthesis pathway of 2-hydroxyisobutyric acid was constructed in Aeromonas hydrophila. It was found that it could synthesize 2-hydroxyisobutyric acid. Then different carbon sources of fatty acids and two-step fermentation were used to optimize the culture conditions. Finally, the yield of 46 mg/L was obtained. This paper provides a preliminary research basis for the synthesis of 2-hydroxyisobutyric acid by microbial method.
【學(xué)位授予單位】：北京化工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R914.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 謝能中;王青艷;朱綺霞;秦艷;陳東;李億;黃日波;;微生物催化合成丙烯酸的研究進(jìn)展[J];廣西科學(xué)院學(xué)報(bào);2013年03期

2 徐誠(chéng),倪兆艾;稀土α-羥基異丁酸配合物的合成與表征[J];杭州大學(xué)學(xué)報(bào)(自然科學(xué)版);1992年02期

，

本文編號(hào)：1477783