基于辅因子调控的S-腺苷蛋氨酸合成研究

发布时间：2018-01-17 13:33

本文关键词：基于辅因子调控的S-腺苷蛋氨酸合成研究　出处：《北京化工大学》2016年博士论文　论文类型：学位论文

【摘要】：S-腺苷蛋氨酸(SAM)是一种在生物中广泛存在并具有重要生理功能的小分子物质。临床上将SAM用于关节炎、肝病、抑郁症等疾病的治疗,效果显著。目前SAM主要通过微生物发酵制备,但存在产量低,前体L-蛋氨酸和ATP供应不足等问题。研究者试图通过代谢工程改造以提高菌株生产SAM的能力,然而SAM在微生物中的代谢途径冗长复杂,而且不是终端代谢产物,仅针对主代谢途径中涉及到的基因进行分子操作,并不能大幅度提高SAM产量。作为胞内重要微环境的辅因子ATP/ADP、NADH/NAD+、 NADPH/NADP+参与了细胞内大量的反应过程,将物质代谢途径联成复杂的网络体系,最终导致物质代谢流的分配受到辅因子形式和浓度的牵制。因此,通过辅因子工程手段调控胞内代谢物的合成具有极大的潜力。本论文以解析辅因子对于SAM合成的调控机理为目的,通过代谢工程策略改造大肠杆菌(Escherichia coli)和酿酒酵母(Saccharomyces cerevisiae)胞内辅因子形式和浓度,综合运用荧光定量PCR和LC/MS/MS方法分析并总结辅因子对于产物合成的生理机制以及能量代谢和其他物质代谢的规律,为高产SAM的菌株的代谢工程改造提供理论基础。主要研究工作如下：1.建立基于合成的sRNA技术用于E. coli体系胞内辅因子调控的系统,通过干扰基因转录水平调控胞内辅因子水平。构建靶向红色荧光蛋白的sRNA结合流式细胞仪和荧光定量PCR,证实了该策略可有效调控胞内基因转录表达,弱化效率可达85%。应用该策略调控副产物竞争消耗ATP和NADPH的代谢途径相关基因,构建了相应的合成sRNA:anti-proB, anti-glnA, anti-argB, anti-aroE, anti-argC, anti-pro A, anti-ilvC和anti-proC。通过荧光定量PCR结果表明目标基因的转录水平都有不同程度的降低。与对照菌相比,带有合成sRNA的重组菌胞内ATP水平和NADPH水平均有所提高,胞内SAM产量提高约1倍。2.在E. coli中通过分别引入NADH激酶(pos5p)以及转氢酶(PntAB)和NAD激酶(YfjB)联用构建两种NADPH再生系统。分析NADPH浓度、NADPH/NADP+比率变化对于胞内物质代谢的扰动及产物SAM合成的关系,发现通过NADPH再生系统提高NADPH水平及NADPH/NADP+比率可以有效地促进胞内SAM的合成。其中基于NADH激酶再生系统使得胞内SAM产量提高13倍,产量达5.30mg/L。3.由于S. cerevisiae中的NADPH在线粒体和细胞质中的代谢相对独立,因此在E. coli的研究基础上,以S. cerevisiae BY4741单倍体模式菌株为研究对象,研究了不同亚细胞结构内NADPH对于产物合成的影响。通过激光共聚焦显微镜证实了成功在S. cerevisiae线粒体中表达了NADH激酶pos5编码),在细胞质中表达了不带信号肽的NADH激酶(pos5△17编码)。实验发现细胞质中NADPH再生的菌株NBYSM-1合成SAM的能力以及NADH/NAD+比率、ATP水平均要明显高于线粒体中NADPH再生的菌株NBYSM-2, NADPH/NADP+比率低于NBYSM-2。说明NADPH对于菌株合成SAM产量有促进作用,ATP对SAM的合成影响更为重要。4.以S. cerevisiae为研究宿主,在过表达sam2的基础上,构建了多种不同形式的ATP调控系统,pRS425-PHXT7-noxE-THXT7,pRS425-PHXT7-vhb-THXT7,pRS425-PHXT7-ptxD-THXT7和pRS425-PHXT7-fdh1-THXT7。结果表明S-腺苷蛋氨酸合成酶活性对于SAM的合成影响极为重要,过表达sam2的菌株胞内SAM产量提高一倍。vhb和ptxD对于胞内SAM的合成有较为显著的促进作用,其中菌株ABYSM-2发酵28 h胞内SAM浓度最高,与对照相比提高67%,可达54.92 mg/L。借助液质结合代谢组分析,发现重组菌株和对照菌株的代谢物差异较大,其中氨基酸途径差异明显,可能是影响SAM合成的重要因素。通过qPCR数据表明NADH水平和ATP水平提高会显著抑制糖酵解及TCA途径关键酶的基因转录水平,其中tdh1, pyk2和idh1受NADH影响较大。其中磷酸戊糖代谢途径的zwf1在ATP水平提高的菌株中没有受到影响,但是受到NADH影响而降低了转录水平。这也导致了碳物质重新分布,从而影响胞内SAM的合成能力。
[Abstract]:S- S-adenosyl-L-methionine (SAM) is a ubiquitous and biological small molecules has important physiological function. Clinically, SAM for arthritis, liver disease, treatment of diseases such as depression, the effect is significant. The SAM is mainly prepared by microbial fermentation, but low yield, former L- methionine and ATP supply insufficient. Researchers tried to improve the ability of strains producing SAM by metabolic engineering, however, the metabolism of SAM in the microbial pathways are lengthy and complex, but not the terminal metabolites, molecular operation only for the main metabolic pathways involved in the gene, and can greatly improve the yield of SAM. As a cofactor of ATP/ADP, intracellular an important micro environment of NADH/NAD+, NADPH/NADP+ in the reaction process of a large number of cells in the metabolic pathways associated with complex network system, resulting in the distribution of material metabolic flux is associated with the Check form and concentration. Therefore, the cofactor engineering means regulating intracellular metabolite synthesis has great potential. In this paper, analysis for the cofactor regulating mechanism of SAM synthesis by metabolic engineering strategies for the purpose of transformation of Escherichia coli (Escherichia coli) and wine yeast (Saccharomyces cerevisiae) cofactor form and concentration inside the cell, using fluorescence quantitative PCR and LC/MS/MS method to analyze and summarize the cofactor for the physiological mechanism of synthesized products and the law of energy metabolism and other metabolic substances, and provide a theoretical basis for the metabolic engineering of high yield SAM strains. The main research work is as follows: 1. to establish a system for the E. coli system of intracellular cofactor synthesis regulation based on sRNA technology, the cofactor level through the regulation of gene transcription level of intracellular interference. Construction of target binding by flow cytometry to red fluorescent protein sRNA Instrument and fluorescent quantitative PCR proved that this method can effectively regulate transcription of intracellular metabolic pathway related gene expression, weakening the efficiency can reach 85%. application of the strategy to control the consumption of ATP and NADPH by-product of competition, construction of the synthesis of sRNA:anti-proB, the corresponding anti-glnA, anti-argB, anti-aroE, anti-argC, anti-pro, A, anti-ilvC and anti-proC. by fluorescence quantitative PCR results showed that the expression level of target gene had been reduced in different degree. Compared with the control bacteria, with the synthesis of sRNA recombinant intracellular ATP level and NADPH level were improved, intracellular SAM production increased about 1 times in.2. E. coli through the introduction of NADH kinase (pos5p) and transhydrogenase (PntAB) and NAD kinase (YfjB) combined with two kinds of construction of NADPH regeneration system. Analysis of NADPH concentration, NADPH/NADP+ ratio changes in the relationship between disturbance of metabolism and intracellular product of SAM synthesis, found through NADPH regeneration system and improve the level of NADPH and NADPH/NADP+ ratio can effectively promote the synthesis of intracellular SAM. NADH kinase regeneration system makes intracellular SAM production increased 13 times on the yield of 5.30mg/L.3. due to S. cerevisiae NADPH in the metabolism of mitochondria and cytoplasm in the phase of independence, therefore based on E. coli. The S. cerevisiae BY4741 haploid model strains as the research object, study the different subcellular structure of NADPH effect on the product synthesis. By laser scanning confocal microscopy confirmed the success of S. cerevisiae in the mitochondria of NADH kinase pos5 encoding), expressed in cytoplasm without signal peptide of NADH kinase (pos5 delta 17 encoding) the ability of strain NBYSM-1. The experimental results showed that synthesis of SAM NADPH regeneration in the cytoplasm and the ratio of NADH/NAD+, ATP levels were significantly higher than in the mitochondria of regenerated NADPH strain N BYSM-2, NADPH/NADP+ NBYSM-2. ratio less than NADPH can promote the synthesis of SAM strain for production, the effect of ATP on Synthesis of SAM.4. in S. cerevisiae is more important for the study of host, in the over expression of Sam2 on the basis of the construction of a variety of ATP control system, different forms of pRS425-PHXT7-noxE-THXT7, pRS425-PHXT7-vhb-THXT7, pRS425-PHXT7-ptxD-THXT7 and pRS425-PHXT7-fdh1-THXT7. results show that the S- Sam synthetase activity is extremely important for the synthesis of SAM, overexpression of Sam2 strain SAM production doubled.Vhb and ptxD have significant effect on the synthesis of intracellular SAM, the strain ABYSM-2 was 28 h and the intracellular SAM concentration is highest, compared with the control group increased by 67%, up to 54.92 mg/L. with the combination of fluid metabonomic analysis, found that the metabolites of recombinant strain and the control strain difference is larger, the difference of amino acid pathway in Ming Dynasty Obviously, might be an important factor affecting the synthesis of SAM. The results indicated that the gene transcription level, NADH level and ATP level increased significantly inhibited glycolysis key enzyme and TCA pathway through which qPCR data tdh1, Pyk2 and IDH1 was affected by NADH. Which was not affected by the pentose phosphate pathway of zwf1 in ATP to improve the level of strain however, the impact of NADH and reduce the level of transcription. This also led to the redistribution of carbon material, thus affecting the intracellular SAM synthesis ability.

【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78;TQ922

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