【摘要】：苯酚是一種非常重要的有機化工原料,廣泛應用于石油化工和生物醫(yī)藥領域。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)苯酚具有廣闊的應用前景。本研究通過克隆苯酚合成基因,質粒過表達苯酚合成途徑中的關鍵酶,構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克隆到表達載體pTrc99A-M上,構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分別克隆到表達載體pACYC184-M上,并檢測了過表達這兩個酶對苯酚產(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鍵酶,過表達之后分別使苯酚產(chǎn)量提高了5.8和68.2倍。為了構建遺傳穩(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Phe009,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進行雙相高細胞密度發(fā)酵,苯酚產(chǎn)量達到了9.5 g/L,轉化率為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.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TQ243.12

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