【摘要】：吲哚丙酮酸(IPA)是很多物質(zhì)的通用中間產(chǎn)物和構(gòu)造組件,這些物質(zhì)在醫(yī)藥和食品行業(yè)中有著重要價(jià)值。IPA同時(shí)也有著一系列廣泛的應(yīng)用,例如作為神經(jīng)系統(tǒng)的治療藥物,護(hù)膚品,以及發(fā)光體�；瘜W(xué)合成IPA是一個(gè)復(fù)雜而且代價(jià)較高的過(guò)程。而利用L-氨基酸氧化酶的生物合成路徑中,其副產(chǎn)物過(guò)氧化氫會(huì)導(dǎo)致IPA的降解。TdiD作為特異性的色氨酸轉(zhuǎn)氨酶,利用其進(jìn)行生物合成可能會(huì)成為高效率的IPA合成的替代方案。大腸桿菌菌株W3110,在含有色氨酸的培養(yǎng)基中,會(huì)有一定量的IPA基礎(chǔ)產(chǎn)量。本研究用W3110作為出發(fā)菌株,通過(guò)代謝工程的方法研究IPA的生物合成。為了提高IPA的產(chǎn)量,實(shí)施了以下的策略:第一步,通過(guò)在W3110菌株中引入密碼子優(yōu)化的tdiD基因,使IPA的水平從41.54±1.26 mg/L上升到52.54±2.08mg/L;第二步,在確認(rèn)了增加苯丙酮酸含量的益處基礎(chǔ)上,構(gòu)建了重組菌株YL03,進(jìn)一步提高IPA的產(chǎn)量。YL03的構(gòu)建是以一個(gè)之前文獻(xiàn)中報(bào)告過(guò)的W3110缺陷型菌株為基礎(chǔ),重組表達(dá)含有不受反饋抑制的DAHP合成酶基因(aroFfbr)和分支酸變位酶/預(yù)苯酸脫水酶基因(pheAfbr)的質(zhì)粒。重組菌株YL03可以積累IPA的數(shù)量達(dá)到158.85±5.36mg/L,這個(gè)產(chǎn)量是野生型W3110菌株的3.82倍;第三步,通過(guò)將Trc啟動(dòng)子更換為一系列不同強(qiáng)度的組成型啟動(dòng)子,并且增加質(zhì)粒的拷貝數(shù),來(lái)對(duì)tdiDco的表達(dá)進(jìn)行優(yōu)化。在這些構(gòu)建的菌株當(dāng)中,YL08的IPA產(chǎn)量最高,可達(dá)到236.42±17.66mg/L。這個(gè)IPA最高產(chǎn)量是野生型W3110產(chǎn)量的5倍以上。最后,本研究評(píng)估了敲除以及過(guò)表達(dá)色氨酸酶基因(tnaA)對(duì)IPA生物合成的影響。敲除tnaA會(huì)引起IPA水平的輕微下降,而過(guò)表達(dá)tnaA則會(huì)造成產(chǎn)量相當(dāng)嚴(yán)重地減少。本論文還對(duì)Terrequinone A在大腸桿菌中的生物合成進(jìn)行了初步探討。Terrequinone A合成通路包括tdiA-tdiE五個(gè)基因,通過(guò)tdiD得到IPA是其中的第一步。利用不同拷貝量的三種質(zhì)粒組合表達(dá)這個(gè)五個(gè)基因,成功構(gòu)建了三個(gè)不同的生產(chǎn)菌株,并進(jìn)行了初步的生物合成研究。本研究闡明了通過(guò)tdiD基因在大腸桿菌中生物合成IPA的可行性。研究得到了一個(gè)IPA的高效率生產(chǎn)菌株YL08,這為以后IPA的合成提供了另一種可能的方式。盡管最終的產(chǎn)量仍然有限,但是本研究論證了IPA的另一種有效且便捷的合成方法。同時(shí)為進(jìn)一步探索Terrequinone A的生物合成奠定了基礎(chǔ)。
[Abstract]:Indole pyruvate (IPA) is a common intermediate and structural component of many substances, which are of great value in the pharmaceutical and food industries. IPA also has a wide range of applications, such as a therapeutic drug for the nervous system, skin care products, And the luminous body. Chemical synthesis of IPA is a complex and costly process. In the biosynthesis pathway of L- amino acid oxidase, hydrogen peroxide, a by-product of L- amino acid oxidase, will lead to the degradation of IPA. As a specific tryptophan transaminase, the biosynthesis of TdiD may be an alternative to the efficient synthesis of IPA. Escherichia coli strain W3110, in the medium containing tryptophan, can produce a certain amount of IPA. In this study, the biosynthesis of IPA was studied by metabolic engineering using W3110 as the starting strain. In order to increase the yield of IPA, the following strategies were implemented: first, by introducing codon optimized tdiD gene into W3110 strain, the level of IPA increased from 41.54 鹵1.26 mg/L to 52.54 鹵2.08 mg / L; the second step, on the basis of confirming the benefits of increasing phenylpyruvate content, The recombinant strain YL03, was constructed to further increase the yield of IPA. The construction of YL03 was based on a W3110 defective strain reported in previous literature. The recombinant plasmid expressed DAHP synthase gene (aroFfbr) and (pheAfbr) gene containing DAHP synthase gene and prephenic acid dehydratase gene which were not inhibited by feedback. The recombinant strain YL03 could accumulate 158.85 鹵5.36 mg / L of IPA, which was 3.82 times higher than that of the wild type W3110. In the third step, the Trc promoter was replaced by a series of component promoters with different intensities, and the copy number of the plasmid was increased. To optimize the expression of tdiDco. Of the constructed strains, YL08 had the highest IPA yield of 236.42 鹵17.66 mg / L. The maximum yield of this IPA is more than five times that of the wild type W3110. Finally, the effects of knockout and overexpression of tryptophan gene (tnaA) on IPA biosynthesis were evaluated. Knockout of tnaA leads to a slight decrease in IPA level, while over-expression of tnaA results in a considerable decrease in output. The biosynthesis of Terrequinone A in Escherichia coli was also discussed in this paper. The. Terrequinone A synthesis pathway includes five tdiA-tdiE genes, and the first step is to obtain IPA by tdiD. The five genes were expressed by using three plasmids with different copy quantities, and three different producing strains were successfully constructed, and preliminary biosynthesis was carried out. This study demonstrated the feasibility of biosynthesis of IPA by tdiD gene in Escherichia coli. A highly efficient strain of IPA, YL08, was obtained, which provides another possible way for the synthesis of IPA in the future. Although the final yield is still limited, this study demonstrates another effective and convenient synthetic method for IPA. At the same time, it lays a foundation for further exploring the biosynthesis of Terrequinone A.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q936

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