本文關(guān)鍵詞：基于輔因子調(diào)控的S-腺苷蛋氨酸合成研究　出處：《北京化工大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： S-腺苷蛋氨酸 輔因子 釀酒酵母 大腸桿菌 NADPH ATP sRNA

【摘要】：S-腺苷蛋氨酸(SAM)是一種在生物中廣泛存在并具有重要生理功能的小分子物質(zhì)。臨床上將SAM用于關(guān)節(jié)炎、肝病、抑郁癥等疾病的治療,效果顯著。目前SAM主要通過(guò)微生物發(fā)酵制備,但存在產(chǎn)量低,前體L-蛋氨酸和ATP供應(yīng)不足等問(wèn)題。研究者試圖通過(guò)代謝工程改造以提高菌株生產(chǎn)SAM的能力,然而SAM在微生物中的代謝途徑冗長(zhǎng)復(fù)雜,而且不是終端代謝產(chǎn)物,僅針對(duì)主代謝途徑中涉及到的基因進(jìn)行分子操作,并不能大幅度提高SAM產(chǎn)量。作為胞內(nèi)重要微環(huán)境的輔因子ATP/ADP、NADH/NAD+、 NADPH/NADP+參與了細(xì)胞內(nèi)大量的反應(yīng)過(guò)程,將物質(zhì)代謝途徑聯(lián)成復(fù)雜的網(wǎng)絡(luò)體系,最終導(dǎo)致物質(zhì)代謝流的分配受到輔因子形式和濃度的牽制。因此,通過(guò)輔因子工程手段調(diào)控胞內(nèi)代謝物的合成具有極大的潛力。本論文以解析輔因子對(duì)于SAM合成的調(diào)控機(jī)理為目的,通過(guò)代謝工程策略改造大腸桿菌(Escherichia coli)和釀酒酵母(Saccharomyces cerevisiae)胞內(nèi)輔因子形式和濃度,綜合運(yùn)用熒光定量PCR和LC/MS/MS方法分析并總結(jié)輔因子對(duì)于產(chǎn)物合成的生理機(jī)制以及能量代謝和其他物質(zhì)代謝的規(guī)律,為高產(chǎn)SAM的菌株的代謝工程改造提供理論基礎(chǔ)。主要研究工作如下：1.建立基于合成的sRNA技術(shù)用于E. coli體系胞內(nèi)輔因子調(diào)控的系統(tǒng),通過(guò)干擾基因轉(zhuǎn)錄水平調(diào)控胞內(nèi)輔因子水平。構(gòu)建靶向紅色熒光蛋白的sRNA結(jié)合流式細(xì)胞儀和熒光定量PCR,證實(shí)了該策略可有效調(diào)控胞內(nèi)基因轉(zhuǎn)錄表達(dá),弱化效率可達(dá)85%。應(yīng)用該策略調(diào)控副產(chǎn)物競(jìng)爭(zhēng)消耗ATP和NADPH的代謝途徑相關(guān)基因,構(gòu)建了相應(yīng)的合成sRNA:anti-proB, anti-glnA, anti-argB, anti-aroE, anti-argC, anti-pro A, anti-ilvC和anti-proC。通過(guò)熒光定量PCR結(jié)果表明目標(biāo)基因的轉(zhuǎn)錄水平都有不同程度的降低。與對(duì)照菌相比,帶有合成sRNA的重組菌胞內(nèi)ATP水平和NADPH水平均有所提高,胞內(nèi)SAM產(chǎn)量提高約1倍。2.在E. coli中通過(guò)分別引入NADH激酶(pos5p)以及轉(zhuǎn)氫酶(PntAB)和NAD激酶(YfjB)聯(lián)用構(gòu)建兩種NADPH再生系統(tǒng)。分析NADPH濃度、NADPH/NADP+比率變化對(duì)于胞內(nèi)物質(zhì)代謝的擾動(dòng)及產(chǎn)物SAM合成的關(guān)系,發(fā)現(xiàn)通過(guò)NADPH再生系統(tǒng)提高NADPH水平及NADPH/NADP+比率可以有效地促進(jìn)胞內(nèi)SAM的合成。其中基于NADH激酶再生系統(tǒng)使得胞內(nèi)SAM產(chǎn)量提高13倍,產(chǎn)量達(dá)5.30mg/L。3.由于S. cerevisiae中的NADPH在線粒體和細(xì)胞質(zhì)中的代謝相對(duì)獨(dú)立,因此在E. coli的研究基礎(chǔ)上,以S. cerevisiae BY4741單倍體模式菌株為研究對(duì)象,研究了不同亞細(xì)胞結(jié)構(gòu)內(nèi)NADPH對(duì)于產(chǎn)物合成的影響。通過(guò)激光共聚焦顯微鏡證實(shí)了成功在S. cerevisiae線粒體中表達(dá)了NADH激酶pos5編碼),在細(xì)胞質(zhì)中表達(dá)了不帶信號(hào)肽的NADH激酶(pos5△17編碼)。實(shí)驗(yàn)發(fā)現(xiàn)細(xì)胞質(zhì)中NADPH再生的菌株NBYSM-1合成SAM的能力以及NADH/NAD+比率、ATP水平均要明顯高于線粒體中NADPH再生的菌株NBYSM-2, NADPH/NADP+比率低于NBYSM-2。說(shuō)明NADPH對(duì)于菌株合成SAM產(chǎn)量有促進(jìn)作用,ATP對(duì)SAM的合成影響更為重要。4.以S. cerevisiae為研究宿主,在過(guò)表達(dá)sam2的基礎(chǔ)上,構(gòu)建了多種不同形式的ATP調(diào)控系統(tǒng),pRS425-PHXT7-noxE-THXT7,pRS425-PHXT7-vhb-THXT7,pRS425-PHXT7-ptxD-THXT7和pRS425-PHXT7-fdh1-THXT7。結(jié)果表明S-腺苷蛋氨酸合成酶活性對(duì)于SAM的合成影響極為重要,過(guò)表達(dá)sam2的菌株胞內(nèi)SAM產(chǎn)量提高一倍。vhb和ptxD對(duì)于胞內(nèi)SAM的合成有較為顯著的促進(jìn)作用,其中菌株ABYSM-2發(fā)酵28 h胞內(nèi)SAM濃度最高,與對(duì)照相比提高67%,可達(dá)54.92 mg/L。借助液質(zhì)結(jié)合代謝組分析,發(fā)現(xiàn)重組菌株和對(duì)照菌株的代謝物差異較大,其中氨基酸途徑差異明顯,可能是影響SAM合成的重要因素。通過(guò)qPCR數(shù)據(jù)表明NADH水平和ATP水平提高會(huì)顯著抑制糖酵解及TCA途徑關(guān)鍵酶的基因轉(zhuǎn)錄水平,其中tdh1, pyk2和idh1受NADH影響較大。其中磷酸戊糖代謝途徑的zwf1在ATP水平提高的菌株中沒(méi)有受到影響,但是受到NADH影響而降低了轉(zhuǎn)錄水平。這也導(dǎo)致了碳物質(zhì)重新分布,從而影響胞內(nèi)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.

【學(xué)位授予單位】：北京化工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;TQ922

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本文編號(hào)：1436466