大肠杆菌中吲哚丙酮酸的代谢工程研究
发布时间：2018-10-21 14:41
【摘要】：吲哚丙酮酸(IPA)是很多物质的通用中间产物和构造组件,这些物质在医药和食品行业中有着重要价值。IPA同时也有着一系列广泛的应用,例如作为神经系统的治疗药物,护肤品,以及发光体。化学合成IPA是一个复杂而且代价较高的过程。而利用L-氨基酸氧化酶的生物合成路径中,其副产物过氧化氢会导致IPA的降解。TdiD作为特异性的色氨酸转氨酶,利用其进行生物合成可能会成为高效率的IPA合成的替代方案。大肠杆菌菌株W3110,在含有色氨酸的培养基中,会有一定量的IPA基础产量。本研究用W3110作为出发菌株,通过代谢工程的方法研究IPA的生物合成。为了提高IPA的产量,实施了以下的策略:第一步,通过在W3110菌株中引入密码子优化的tdiD基因,使IPA的水平从41.54±1.26 mg/L上升到52.54±2.08mg/L;第二步,在确认了增加苯丙酮酸含量的益处基础上,构建了重组菌株YL03,进一步提高IPA的产量。YL03的构建是以一个之前文献中报告过的W3110缺陷型菌株为基础,重组表达含有不受反馈抑制的DAHP合成酶基因(aroFfbr)和分支酸变位酶/预苯酸脱水酶基因(pheAfbr)的质粒。重组菌株YL03可以积累IPA的数量达到158.85±5.36mg/L,这个产量是野生型W3110菌株的3.82倍;第三步,通过将Trc启动子更换为一系列不同强度的组成型启动子,并且增加质粒的拷贝数,来对tdiDco的表达进行优化。在这些构建的菌株当中,YL08的IPA产量最高,可达到236.42±17.66mg/L。这个IPA最高产量是野生型W3110产量的5倍以上。最后,本研究评估了敲除以及过表达色氨酸酶基因(tnaA)对IPA生物合成的影响。敲除tnaA会引起IPA水平的轻微下降,而过表达tnaA则会造成产量相当严重地减少。本论文还对Terrequinone A在大肠杆菌中的生物合成进行了初步探讨。Terrequinone A合成通路包括tdiA-tdiE五个基因,通过tdiD得到IPA是其中的第一步。利用不同拷贝量的三种质粒组合表达这个五个基因,成功构建了三个不同的生产菌株,并进行了初步的生物合成研究。本研究阐明了通过tdiD基因在大肠杆菌中生物合成IPA的可行性。研究得到了一个IPA的高效率生产菌株YL08,这为以后IPA的合成提供了另一种可能的方式。尽管最终的产量仍然有限,但是本研究论证了IPA的另一种有效且便捷的合成方法。同时为进一步探索Terrequinone A的生物合成奠定了基础。
[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 卤1.26 mg/L to 52.54 卤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 卤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 卤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.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936
本文编号：2285411
[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 卤1.26 mg/L to 52.54 卤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 卤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 卤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.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q936
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