【摘要】：莽草酸(Shikimic acid,SA)作為抗禽流感特效藥達(dá)菲的關(guān)鍵起始原料而備受關(guān)注,其衍生物具有抗腫瘤、防止血栓形成等多種功效。白藜蘆醇(Resveratrol,Res)及其衍生物具有抗腫瘤、延緩衰老及保護(hù)心腦血管等多種功效,在食品和營養(yǎng)補(bǔ)充劑行業(yè)應(yīng)用廣泛。目前這兩種化合物的生產(chǎn)都主要從植物中提取,對植物資源依賴嚴(yán)重。微生物具有生長代謝快、操作簡便等多種優(yōu)點(diǎn),越來越多的研究者通過微生物來合成植物來源的重要代謝產(chǎn)物。然而目前的研究大都局限于利用質(zhì)粒表達(dá)載體來強(qiáng)化相關(guān)基因,這種方法有其自身缺陷。近年來基因定點(diǎn)整合技術(shù)由于其穩(wěn)定性及良好效果而受到研究者的重視。本研究基于合成生物學(xué)原理,首次利用基因定點(diǎn)整合技術(shù)改造大腸桿菌BW25113構(gòu)建SA高產(chǎn)菌株及Res從頭合成重組菌株。對于SA生產(chǎn)菌株的構(gòu)建:(1)敲除下游莽草酸激酶基因aro L和aro K,構(gòu)建四基因共表達(dá)質(zhì)粒p ETDuet-GBAE,使SA合成的四個關(guān)鍵酶Aro G(3-脫氧-阿拉伯庚酮糖酸-7-磷酸合成酶)、Aro B(3-脫氫奎尼酸合成酶)、Tkt A(轉(zhuǎn)酮醇酶I)和Aro E(SA脫氫酶)以協(xié)同效應(yīng)的方式促進(jìn)SA的生產(chǎn),SA產(chǎn)量達(dá)到1.08 g/L;(2)首次嘗試通過基因定點(diǎn)整合的方式將上述四個基因定點(diǎn)整合入磷酸烯醇式丙酮酸糖磷酸轉(zhuǎn)移酶系統(tǒng)(PTS)基因pts HIcrr位點(diǎn),在降低前體磷酸烯醇式丙酮酸(PEP)消耗的同時促進(jìn)SA的合成,SA產(chǎn)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提高葡萄糖轉(zhuǎn)運(yùn)速率,縮短發(fā)酵周期;(4)將PEP合成酶基因pps A整合入代謝調(diào)控基因tyr R位點(diǎn)進(jìn)一步增加PEP的供應(yīng),所構(gòu)建的重組菌株SA5可生產(chǎn)SA 2.83 g/L,是出發(fā)菌株BW25113(?aroL/aro K,DE3)的94倍;(5)添加質(zhì)粒p ETDuet-GBAE再次過表達(dá)關(guān)鍵基因,SA產(chǎn)量提高到4.14 g/L,生產(chǎn)率為23.56%;(6)在發(fā)酵罐放大培養(yǎng)中,首次嘗試使用葡萄糖和甘油混合碳源進(jìn)行發(fā)酵,達(dá)到生產(chǎn)速率與持續(xù)生產(chǎn)的雙贏。最終SA產(chǎn)量可達(dá)到27.41 g/L,單位時間產(chǎn)率為0.57g/L·h,是葡萄糖單一碳源的兩倍。對于Res生產(chǎn)菌株的構(gòu)建:(1)將來源于葡萄(Vitis vinifera)的二苯乙烯合酶基因sts定點(diǎn)整合入tyr R基因位點(diǎn),來源于黏紅酵母(Rhodotorula glutinis)的酪氨酸解氨酶基因tal和來源于荷蘭芹(Petroselinum crispum)的4-香豆酸Co A連接酶基因4cl定點(diǎn)整合入色氨酸合成相關(guān)基因trp ED位點(diǎn),首次構(gòu)建真正意義上可利用葡萄糖從頭合成Res的重組大腸桿菌Res1,其Res產(chǎn)量達(dá)到4.61 mg/L;(2)將來源于三葉草根瘤菌(Rhizobium trifolii)的丙二酰Co A合成基因mat B和丙二酸鹽載體蛋白基因mat C定點(diǎn)整合入苯丙氨酸合成相關(guān)基因phe LA位點(diǎn)以增加丙二酰Co A的供應(yīng),Res產(chǎn)量提高到9.35 mg/L;(3)將SA合成關(guān)鍵基因(aro G、aro B、tkt A、aro E)及葡萄糖轉(zhuǎn)運(yùn)基因glk和gal P定點(diǎn)整合入pts HIcrr位點(diǎn)以強(qiáng)化上游代謝途徑基因,Res產(chǎn)量增加到11.41 mg/L;(4)通過質(zhì)粒強(qiáng)化Res合成的上述外源基因后Res產(chǎn)量又進(jìn)一步提高到13.70 mg/L,是菌株Res1的近3倍。(5)在后續(xù)研究中嘗試添加底物酪氨酸或?qū)ο愣顾?p-CA),結(jié)果發(fā)酵液中出現(xiàn)了兩個副產(chǎn)物Res-der1和Res-der2,它們均是p-CA的衍生物。其中Res-der2是p-CA脫羧后的產(chǎn)物4-乙烯基苯酚,其含量隨著底物p-CA添加量的增加而增加。據(jù)此推測在合成Res的過程中,中間產(chǎn)物p-CA會被脫羧或轉(zhuǎn)化為其它化合物,阻礙4-香豆酰Co A的合成,最終影響Res的進(jìn)一步生產(chǎn)。總之,本研究利用合成生物學(xué)方法,首次通過基因定點(diǎn)整合的方式對相關(guān)基因進(jìn)行過表達(dá),成功構(gòu)建SA高產(chǎn)菌株及真正意義上從頭合成的Res生產(chǎn)菌株,為以后的研究奠定了基礎(chǔ)。所構(gòu)建的重組菌株可組成型表達(dá)相關(guān)基因,在發(fā)酵過程中無需添加誘導(dǎo)劑IPTG及抗生素,具有操作簡便、環(huán)境友好、適于工業(yè)化生產(chǎn)等優(yōu)點(diǎn)。
[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.
【學(xué)位授予單位】：中國醫(yī)藥工業(yè)研究總院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R915
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本文編號：2290869