产苯酚大肠杆菌细胞工厂的构建

发布时间：2019-03-18 14:11

【摘要】：苯酚是一种非常重要的有机化工原料,广泛应用于石油化工和生物医药领域。目前,全球97%以上的苯酚是通过异丙苯法生产的。异丙苯法具有污染严重、危险性高等缺点。以绿色环保的微生物发酵法生产苯酚具有广阔的应用前景。本研究通过克隆苯酚合成基因,质粒过表达苯酚合成途径中的关键酶,构建出了新型的苯酚生物合成途径并鉴定出了该途径中的关键酶。然后通过整合ycl操纵子、定点突变并调控aroG基因、整合ubiC基因和调控ycl操纵子,得到了苯酚产量较高的大肠杆菌细胞工厂。首先,将来源于E. coli W (ATCC 9637)的对羟基苯甲酸脱羧酶基因簇yclBCD克隆到表达载体pTrc99A-M上,构建出了苯酚生物合成途径；然后将编码3-脱氧-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(突变后的aroG基因)和ubiC分别克隆到表达载体pACYC184-M上,并检测了过表达这两个酶对苯酚产量的影响。研究发现这两个酶都是苯酚合成途径中的关键酶,过表达之后分别使苯酚产量提高了5.8和68.2倍。为了构建遗传稳定的苯酚生产菌株,将ycl操纵子整合到野生型大肠杆菌ATCC8739染色体的idhA(编码乳酸脱氢酶)位点,得到了苯酚产量为1.7 mg/L的起始菌株；然后定点突变aroG基因,并进行染色体水平的调控,解除DAHP合酶的反馈抑制,使苯酚产量提高了4.3倍；随后在pflB(编码丙酮酸甲酸裂解酶)位点整合了ATCC8739的ubiC基因(记做p-ubiC基因),并使用不同强度启动子调控p-ubiC基因来提高对羟基苯甲酸(pHBA)的供给,效果最好的菌株的苯酚产量提高了19.2倍；之后使用不同强度启动子调控ycl操纵子,得到苯酚产量最高的重组菌株Phe009,苯酚产量又提高了36%,产量为249.9 mg/L。最后,以三丁酸甘油酯为苯酚萃取剂,将Phe009进行双相高细胞密度发酵,苯酚产量达到了9.5 g/L,转化率为0.061 g苯酚儋葡萄糖。通过整合对羟基苯甲酸脱羧酶、解除DAHP合酶的反馈抑制、提高前体物质pHBA的供给和提高脱羧酶的活性,得到了一株遗传稳定、苯酚产量较高的菌株,相对于起始菌株,苯酚产量提高了147倍。通过双相高密度发酵,苯酚产量达到了9.5 g/L,迄今为止,这是运用代谢工程手段改造大肠杆菌获得的苯酚产量最高的菌株。
[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.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ243.12

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