發(fā)布時間：2019-03-18 14:11

【摘要】：苯酚是一種非常重要的有機化工原料,廣泛應(yīng)用于石油化工和生物醫(yī)藥領(lǐng)域。目前,全球97%以上的苯酚是通過異丙苯法生產(chǎn)的。異丙苯法具有污染嚴(yán)重、危險性高等缺點。以綠色環(huán)保的微生物發(fā)酵法生產(chǎn)苯酚具有廣闊的應(yīng)用前景。本研究通過克隆苯酚合成基因,質(zhì)粒過表達(dá)苯酚合成途徑中的關(guān)鍵酶,構(gòu)建出了新型的苯酚生物合成途徑并鑒定出了該途徑中的關(guān)鍵酶。然后通過整合ycl操縱子、定點突變并調(diào)控aroG基因、整合ubiC基因和調(diào)控ycl操縱子,得到了苯酚產(chǎn)量較高的大腸桿菌細(xì)胞工廠。首先,將來源于E. coli W (ATCC 9637)的對羥基苯甲酸脫羧酶基因簇yclBCD克隆到表達(dá)載體pTrc99A-M上,構(gòu)建出了苯酚生物合成途徑；然后將編碼3-脫氧-D-阿拉伯庚酮糖-7-磷酸(DAHP)合酶和分支酸裂解酶(UbiC)的基因aroGfbr(突變后的aroG基因)和ubiC分別克隆到表達(dá)載體pACYC184-M上,并檢測了過表達(dá)這兩個酶對苯酚產(chǎn)量的影響。研究發(fā)現(xiàn)這兩個酶都是苯酚合成途徑中的關(guān)鍵酶,過表達(dá)之后分別使苯酚產(chǎn)量提高了5.8和68.2倍。為了構(gòu)建遺傳穩(wěn)定的苯酚生產(chǎn)菌株,將ycl操縱子整合到野生型大腸桿菌ATCC8739染色體的idhA(編碼乳酸脫氫酶)位點,得到了苯酚產(chǎn)量為1.7 mg/L的起始菌株；然后定點突變aroG基因,并進行染色體水平的調(diào)控,解除DAHP合酶的反饋抑制,使苯酚產(chǎn)量提高了4.3倍；隨后在pflB(編碼丙酮酸甲酸裂解酶)位點整合了ATCC8739的ubiC基因(記做p-ubiC基因),并使用不同強度啟動子調(diào)控p-ubiC基因來提高對羥基苯甲酸(pHBA)的供給,效果最好的菌株的苯酚產(chǎn)量提高了19.2倍；之后使用不同強度啟動子調(diào)控ycl操縱子,得到苯酚產(chǎn)量最高的重組菌株P(guān)he009,苯酚產(chǎn)量又提高了36%,產(chǎn)量為249.9 mg/L。最后,以三丁酸甘油酯為苯酚萃取劑,將Phe009進行雙相高細(xì)胞密度發(fā)酵,苯酚產(chǎn)量達(dá)到了9.5 g/L,轉(zhuǎn)化率為0.061 g苯酚儋葡萄糖。通過整合對羥基苯甲酸脫羧酶、解除DAHP合酶的反饋抑制、提高前體物質(zhì)pHBA的供給和提高脫羧酶的活性,得到了一株遺傳穩(wěn)定、苯酚產(chǎn)量較高的菌株,相對于起始菌株,苯酚產(chǎn)量提高了147倍。通過雙相高密度發(fā)酵,苯酚產(chǎn)量達(dá)到了9.5 g/L,迄今為止,這是運用代謝工程手段改造大腸桿菌獲得的苯酚產(chǎn)量最高的菌株。
[Abstract]:Phenol is a very important organic chemical raw material, widely used in petrochemical and biomedical fields. At present, more than 97% of phenol in the world is produced by cumene process. Cumene method has the disadvantage of serious pollution and high risk. The production of phenol by green and environmental-friendly microbial fermentation has a broad application prospect. In this study, a novel phenol biosynthesis pathway was constructed by cloning the phenol synthesis gene and overexpressing the key enzymes in the phenol synthesis pathway, and the key enzymes in the pathway were identified. Then, by integrating ycl operon, site-directed mutation and regulation of aroG gene, integrating ubiC gene and regulating ycl operon, a high phenol production Escherichia coli cell factory was obtained. Firstly, the p-hydroxybenzoic acid decarboxylase gene cluster yclBCD derived from E. coli W (ATCC 9637 was cloned into the expression vector pTrc99A-M to construct a phenol biosynthesis pathway. Then the genes aroGfbr (mutated aroG gene) and ubiC encoding 3-deoxy-D-arabinose-7-phosphate (DAHP) synthase and branched acid lyase (UbiC) were cloned into the expression vector pACYC184-M, respectively. The effects of over-expression of these two enzymes on phenol production were also examined. It was found that these two enzymes were the key enzymes in phenol synthesis pathway, and the yield of phenol was increased by 5.8 and 68.2 times respectively after over-expression. In order to construct a genetically stable phenol producing strain, the ycl operon was integrated into the idhA (encoding lactate dehydrogenase) site of ATCC8739 chromosome of wild-type Escherichia coli, and the initial strain with phenol yield of 1.7 mg/L was obtained. Then the site-directed mutation of aroG gene and the regulation of chromosome level were carried out to relieve the feedback inhibition of DAHP synthase and increase the yield of phenol by 4.3 times. Then the ubiC gene of ATCC8739 (known as p-ubiC gene) was integrated into the pflB site, and the p-ubiC gene was regulated by different intensity promoters to improve the supply of (pHBA) to p-hydroxybenzoic acid. The phenol yield of the best strain was increased by 19.2 times. Then using different intensity promoters to control the ycl operon, the recombinant strain Phe009, which produced the highest phenol yield, increased the yield of phenol by 36%, and the yield was 249.9 mg/L.. Finally, the yield of phenol was 9.5g / L and the conversion rate was 0.061 g phenol glucose by using glycerol tributyrate as phenol extractant to ferment Phe009 with two-phase high cell density. By integrating p-hydroxybenzoic acid decarboxylase, releasing the feedback inhibition of DAHP synthase, increasing the supply of precursor pHBA and increasing the activity of decarboxylase, a strain with high yield of phenol and stable heredity was obtained. Phenol production increased 147 times. The yield of phenol reached 9.5 g / L through biphasic high-density fermentation. So far, it is the highest strain of phenol production in E. coli by means of metabolic engineering.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TQ243.12

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 蘇國富,H.N.Brahmbhatt,K.N.Timmis;志賀氏毒素B亞單位在大腸桿菌細(xì)胞表面表達(dá)的研究[J];中國科學(xué)(B輯 化學(xué) 生命科學(xué) 地學(xué));1993年01期

2 羅少華;邵偉;仇敏;;包埋-交聯(lián)法固定大腸桿菌細(xì)胞制備γ-氨基丁酸的研究[J];中國釀造;2011年11期

3 左曉佳;牛衛(wèi)寧;田林奇;欽傳光;;大腸桿菌細(xì)胞固定化方法研究進展[J];化學(xué)與生物工程;2010年04期

4 ;一種蛋白可使細(xì)胞分裂停止而生長繼續(xù)[J];中國食品學(xué)報;2013年01期

5 趙景聯(lián);;固定化大腸桿菌細(xì)胞生產(chǎn)γ-氨基丁酸的研究[J];生物工程學(xué)報;1989年02期

6 居乃琥,仇昌明,黃國英,馬雯,金蕾;用明膠-戊二醛法固定大腸桿菌細(xì)胞生產(chǎn)L-天門冬氨酸[J];微生物學(xué)通報;1983年01期

7 蔡潤生,武濟民;聚醚抗生素W341的生化和生物性質(zhì)[J];抗生素;1985年06期

8 劉志洪,李文化,沈萍,蔡汝秀;Fura-2熒光探針研究Ca~(2+)對大腸桿菌細(xì)胞的跨膜作用[J];化學(xué)學(xué)報;2004年04期

9 寇秀芬,王禎祥;三醋酸纖維素包埋產(chǎn)青霉素�；�酶的大腸桿菌細(xì)胞[J];微生物學(xué)通報;1984年04期

10 楊秦;劉會雪;李勤;楊曉達(dá);王夔;;核磁共振法研究大腸桿菌細(xì)胞內(nèi)La~(3+)與鈣調(diào)蛋白的相互作用[J];中國稀土學(xué)報;2007年03期

相關(guān)會議論文 前1條

1 盧群;丘泰球;羅登林;黃卓烈;;超聲物理效應(yīng)對大腸桿菌細(xì)胞膜的影響[A];2005年全國超聲醫(yī)學(xué)工程學(xué)術(shù)會議論文集[C];2005年

相關(guān)重要報紙文章 前3條

1 劉海英;細(xì)菌變生物工廠,僅用3天[N];科技日報;2009年

2 記者 桂運安;國際基因工程機器大賽 中國科技大學(xué)摘兩金[N];安徽日報;2010年

3 錢錚;耐藥病菌細(xì)胞膜上存在“水泵”蛋白[N];醫(yī)藥經(jīng)濟報;2006年

相關(guān)博士學(xué)位論文 前1條

1 葉炎生;細(xì)胞內(nèi)蛋白質(zhì)的~(19)F核磁共振方法與應(yīng)用研究[D];中國科學(xué)院研究生院(武漢物理與數(shù)學(xué)研究所);2015年

相關(guān)碩士學(xué)位論文 前3條

1 苗良田;產(chǎn)苯酚大腸桿菌細(xì)胞工廠的構(gòu)建[D];天津科技大學(xué);2015年

2 劉峰;AspC介導(dǎo)的天冬氨酸代謝協(xié)調(diào)大腸桿菌細(xì)胞周期[D];內(nèi)蒙古大學(xué);2014年

3 徐麗香;細(xì)菌脂肪酶在大腸桿菌細(xì)胞表面的功能性展示[D];福建師范大學(xué);2014年

，

本文編號：2442940