合成生物学技术改造大肠杆菌生产莽草酸及白藜芦醇
发布时间：2018-10-24 08:30
【摘要】：莽草酸(Shikimic acid,SA)作为抗禽流感特效药达菲的关键起始原料而备受关注,其衍生物具有抗肿瘤、防止血栓形成等多种功效。白藜芦醇(Resveratrol,Res)及其衍生物具有抗肿瘤、延缓衰老及保护心脑血管等多种功效,在食品和营养补充剂行业应用广泛。目前这两种化合物的生产都主要从植物中提取,对植物资源依赖严重。微生物具有生长代谢快、操作简便等多种优点,越来越多的研究者通过微生物来合成植物来源的重要代谢产物。然而目前的研究大都局限于利用质粒表达载体来强化相关基因,这种方法有其自身缺陷。近年来基因定点整合技术由于其稳定性及良好效果而受到研究者的重视。本研究基于合成生物学原理,首次利用基因定点整合技术改造大肠杆菌BW25113构建SA高产菌株及Res从头合成重组菌株。对于SA生产菌株的构建:(1)敲除下游莽草酸激酶基因aro L和aro K,构建四基因共表达质粒p ETDuet-GBAE,使SA合成的四个关键酶Aro G(3-脱氧-阿拉伯庚酮糖酸-7-磷酸合成酶)、Aro B(3-脱氢奎尼酸合成酶)、Tkt A(转酮醇酶I)和Aro E(SA脱氢酶)以协同效应的方式促进SA的生产,SA产量达到1.08 g/L;(2)首次尝试通过基因定点整合的方式将上述四个基因定点整合入磷酸烯醇式丙酮酸糖磷酸转移酶系统(PTS)基因pts HIcrr位点,在降低前体磷酸烯醇式丙酮酸(PEP)消耗的同时促进SA的合成,SA产量提高到2.08 g/L;(3)整合入半乳糖透性酶基因gal P和葡糖激酶基因glk提高葡萄糖转运速率,缩短发酵周期;(4)将PEP合成酶基因pps A整合入代谢调控基因tyr R位点进一步增加PEP的供应,所构建的重组菌株SA5可生产SA 2.83 g/L,是出发菌株BW25113(?aroL/aro K,DE3)的94倍;(5)添加质粒p ETDuet-GBAE再次过表达关键基因,SA产量提高到4.14 g/L,生产率为23.56%;(6)在发酵罐放大培养中,首次尝试使用葡萄糖和甘油混合碳源进行发酵,达到生产速率与持续生产的双赢。最终SA产量可达到27.41 g/L,单位时间产率为0.57g/L·h,是葡萄糖单一碳源的两倍。对于Res生产菌株的构建:(1)将来源于葡萄(Vitis vinifera)的二苯乙烯合酶基因sts定点整合入tyr R基因位点,来源于黏红酵母(Rhodotorula glutinis)的酪氨酸解氨酶基因tal和来源于荷兰芹(Petroselinum crispum)的4-香豆酸Co A连接酶基因4cl定点整合入色氨酸合成相关基因trp ED位点,首次构建真正意义上可利用葡萄糖从头合成Res的重组大肠杆菌Res1,其Res产量达到4.61 mg/L;(2)将来源于三叶草根瘤菌(Rhizobium trifolii)的丙二酰Co A合成基因mat B和丙二酸盐载体蛋白基因mat C定点整合入苯丙氨酸合成相关基因phe LA位点以增加丙二酰Co A的供应,Res产量提高到9.35 mg/L;(3)将SA合成关键基因(aro G、aro B、tkt A、aro E)及葡萄糖转运基因glk和gal P定点整合入pts HIcrr位点以强化上游代谢途径基因,Res产量增加到11.41 mg/L;(4)通过质粒强化Res合成的上述外源基因后Res产量又进一步提高到13.70 mg/L,是菌株Res1的近3倍。(5)在后续研究中尝试添加底物酪氨酸或对香豆酸(p-CA),结果发酵液中出现了两个副产物Res-der1和Res-der2,它们均是p-CA的衍生物。其中Res-der2是p-CA脱羧后的产物4-乙烯基苯酚,其含量随着底物p-CA添加量的增加而增加。据此推测在合成Res的过程中,中间产物p-CA会被脱羧或转化为其它化合物,阻碍4-香豆酰Co A的合成,最终影响Res的进一步生产。总之,本研究利用合成生物学方法,首次通过基因定点整合的方式对相关基因进行过表达,成功构建SA高产菌株及真正意义上从头合成的Res生产菌株,为以后的研究奠定了基础。所构建的重组菌株可组成型表达相关基因,在发酵过程中无需添加诱导剂IPTG及抗生素,具有操作简便、环境友好、适于工业化生产等优点。
[Abstract]:Shikimic acid (SA) is a key starting material for resisting avian influenza, and its derivatives have many functions such as anti-tumor, prevention of thrombosis and so on. Resvertrol, Res. and its derivatives have many efficacies of resisting tumor, delaying aging and protecting cardiovascular and cerebrovascular diseases, and has wide application in food and nutritional supplement industry. At present, the production of both compounds is mainly extracted from plants, and the dependence of plant resources is severe. Microbes have many advantages such as rapid growth and metabolism, simple and convenient operation, and more and more researchers synthesize important metabolites of plant origin through micro-organisms. However, the current research is mostly limited to using plasmid expression vector to strengthen the related gene, and the method has its own defect. In recent years, gene-directed mutagenesis has been paid more attention by researchers due to its stability and good results. Based on the principle of synthetic biology, the high-yield strain of SA and the recombinant strain of Res de novo were constructed by using gene-directed mutagenesis technique to transform E. coli JM25113 for the first time. For the construction of SA production strains: (1) knock out the downstream shikimate kinase gene BamL and LacK, construct the four-gene co-expression plasmid p ETDuet-GBAE, make the four key enzymes Aro G (3-desoxy-arabidonic acid-7-phosphate synthase) synthesized by SA, Aro B (3-dehydroquinic acid synthase), Tkt A (ketolase I) and Aro E (SA dehydrogenase) promoted SA production in a synergistic way, SA yield reached 1.08 g/ L; (2) the first attempt to fix the above four gene sites into the phosphoenol pyruvate phosphotransferase system (PTS) gene pts HIcrr site by site-directed mutagenesis, while promoting the synthesis of the SA while reducing the consumption of the precursor phosphoenol pyruvate (PEP), the yield of the SA is increased to 2.08 g/ L; (3) the whole combination of the galacturizing enzyme gene PpP and the glucokinase gene glk improves the glucose transport rate and shortens the fermentation period; and (4) the PEP synthetase gene pps A is integrated into the metabolic regulation gene tyr R site to further increase the supply of PEP, The constructed recombinant strain SA5 can produce SA 2.83 g/ L, which is 94 times that of the starting strain ATCC 25113 (? aroL/ LacK, DE3); (5) the addition of the plasmid p ETDuet-GBAE once again overexpresses the key gene, the SA yield is increased to 4.14g/ L, the productivity is 23.56%, and (6) in the amplification culture of the fermentation tank, The first attempt was to use glucose and glycerol mixed carbon sources for fermentation to achieve a win-win of production rate and continuous production. The final SA yield can reach 27.41g/ L, and the yield per unit time is 0. 57g/ L 路 h, which is twice the single carbon source of glucose. For the construction of the Res-producing strain: (1) a two-styrene synthase gene sts derived from Vitis Vinifera is integrated into a tyr R gene site, The tyrosine deammoniase gene (t) derived from Rhodotorula glutinis and the 4-cedar-acid Co A ligase gene 4cl derived from the Netherlands celery (Perosinum crispum) are integrated into the tryptophan synthesis-related gene trp ED site, For the first time, the recombinant E. coli Res1 can be used to synthesize Res1 in real sense, and its Res yield reached 4.61 mg/ L. (2) synthesizing the gene mat B and the acid salt carrier protein gene mat C, which are derived from Rhizobium trifolii, into phenylalanine to synthesize the related gene phe LA site to increase the supply of the C-2 and Co A, and the Res yield is increased to 9. 35mg/ L; (3) The key genes of SA were integrated into the pts HIcrr site to strengthen the upstream metabolic pathway gene, and the Res yield increased to 11.41 mg/ L. (4) The Res yield was further increased to 13. 70mg/ L after the exogenous gene was strengthened by plasmid-enhanced Res, which was nearly 3 times that of strain Res1. (5) In the follow-up study, we tried to add substrate tyrosine or p-CA, resulting in two by-products Res-der1 and Res-der2, both of which were the derivatives of p-CA. where Res-der2 is the product 4-vinylphenol after p-CA affinity, whose content increases as the amount of substrate p-CA increases. Therefore, in the process of synthesizing Res, the intermediate product p-CA can be converted into other compounds, hindering the synthesis of 4-Xiangdou-Co A and finally affecting the further production of Res. In conclusion, using synthetic biology method, the gene was expressed for the first time by gene-directed mutagenesis, the high-yield strain of SA was successfully constructed and the real-time synthetic Res-producing strain was successfully constructed, which laid the foundation for later research. The constructed recombinant strain can form constitutive expression related genes, and induction and antibiotics are not required to be added in the fermentation process, and the recombinant strain has the advantages of simple operation, environmental friendliness, suitability for industrial production and the like.
【学位授予单位】：中国医药工业研究总院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R915
，
本文编号：2290869
[Abstract]:Shikimic acid (SA) is a key starting material for resisting avian influenza, and its derivatives have many functions such as anti-tumor, prevention of thrombosis and so on. Resvertrol, Res. and its derivatives have many efficacies of resisting tumor, delaying aging and protecting cardiovascular and cerebrovascular diseases, and has wide application in food and nutritional supplement industry. At present, the production of both compounds is mainly extracted from plants, and the dependence of plant resources is severe. Microbes have many advantages such as rapid growth and metabolism, simple and convenient operation, and more and more researchers synthesize important metabolites of plant origin through micro-organisms. However, the current research is mostly limited to using plasmid expression vector to strengthen the related gene, and the method has its own defect. In recent years, gene-directed mutagenesis has been paid more attention by researchers due to its stability and good results. Based on the principle of synthetic biology, the high-yield strain of SA and the recombinant strain of Res de novo were constructed by using gene-directed mutagenesis technique to transform E. coli JM25113 for the first time. For the construction of SA production strains: (1) knock out the downstream shikimate kinase gene BamL and LacK, construct the four-gene co-expression plasmid p ETDuet-GBAE, make the four key enzymes Aro G (3-desoxy-arabidonic acid-7-phosphate synthase) synthesized by SA, Aro B (3-dehydroquinic acid synthase), Tkt A (ketolase I) and Aro E (SA dehydrogenase) promoted SA production in a synergistic way, SA yield reached 1.08 g/ L; (2) the first attempt to fix the above four gene sites into the phosphoenol pyruvate phosphotransferase system (PTS) gene pts HIcrr site by site-directed mutagenesis, while promoting the synthesis of the SA while reducing the consumption of the precursor phosphoenol pyruvate (PEP), the yield of the SA is increased to 2.08 g/ L; (3) the whole combination of the galacturizing enzyme gene PpP and the glucokinase gene glk improves the glucose transport rate and shortens the fermentation period; and (4) the PEP synthetase gene pps A is integrated into the metabolic regulation gene tyr R site to further increase the supply of PEP, The constructed recombinant strain SA5 can produce SA 2.83 g/ L, which is 94 times that of the starting strain ATCC 25113 (? aroL/ LacK, DE3); (5) the addition of the plasmid p ETDuet-GBAE once again overexpresses the key gene, the SA yield is increased to 4.14g/ L, the productivity is 23.56%, and (6) in the amplification culture of the fermentation tank, The first attempt was to use glucose and glycerol mixed carbon sources for fermentation to achieve a win-win of production rate and continuous production. The final SA yield can reach 27.41g/ L, and the yield per unit time is 0. 57g/ L 路 h, which is twice the single carbon source of glucose. For the construction of the Res-producing strain: (1) a two-styrene synthase gene sts derived from Vitis Vinifera is integrated into a tyr R gene site, The tyrosine deammoniase gene (t) derived from Rhodotorula glutinis and the 4-cedar-acid Co A ligase gene 4cl derived from the Netherlands celery (Perosinum crispum) are integrated into the tryptophan synthesis-related gene trp ED site, For the first time, the recombinant E. coli Res1 can be used to synthesize Res1 in real sense, and its Res yield reached 4.61 mg/ L. (2) synthesizing the gene mat B and the acid salt carrier protein gene mat C, which are derived from Rhizobium trifolii, into phenylalanine to synthesize the related gene phe LA site to increase the supply of the C-2 and Co A, and the Res yield is increased to 9. 35mg/ L; (3) The key genes of SA were integrated into the pts HIcrr site to strengthen the upstream metabolic pathway gene, and the Res yield increased to 11.41 mg/ L. (4) The Res yield was further increased to 13. 70mg/ L after the exogenous gene was strengthened by plasmid-enhanced Res, which was nearly 3 times that of strain Res1. (5) In the follow-up study, we tried to add substrate tyrosine or p-CA, resulting in two by-products Res-der1 and Res-der2, both of which were the derivatives of p-CA. where Res-der2 is the product 4-vinylphenol after p-CA affinity, whose content increases as the amount of substrate p-CA increases. Therefore, in the process of synthesizing Res, the intermediate product p-CA can be converted into other compounds, hindering the synthesis of 4-Xiangdou-Co A and finally affecting the further production of Res. In conclusion, using synthetic biology method, the gene was expressed for the first time by gene-directed mutagenesis, the high-yield strain of SA was successfully constructed and the real-time synthetic Res-producing strain was successfully constructed, which laid the foundation for later research. The constructed recombinant strain can form constitutive expression related genes, and induction and antibiotics are not required to be added in the fermentation process, and the recombinant strain has the advantages of simple operation, environmental friendliness, suitability for industrial production and the like.
【学位授予单位】：中国医药工业研究总院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R915
，
本文编号：2290869
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2290869.html
最近更新
教材专著
