本文選題：氮代謝阻遏 + 精氨酸代謝��； 參考：《江南大學(xué)》2017年博士論文

【摘要】：氨基甲酸乙酯是一種廣泛存在于發(fā)酵食品中的有害物質(zhì),它的存在嚴重影響了我國傳統(tǒng)發(fā)酵食品的安全性。尿素是黃酒中氨基甲酸乙酯生成最重要的前體物質(zhì)之一,降低發(fā)酵末期黃酒中尿素的含量對于黃酒中氨基甲酸乙酯含量的降低具有重要意義。本文通過模式菌株研究氮代謝阻遏調(diào)控因子、精氨酸轉(zhuǎn)運途徑和尿素轉(zhuǎn)運蛋白Dur3p泛素化對釀酒酵母發(fā)酵末期尿素生成的影響,釀酒酵母整體核小體排布與氮代謝阻遏相關(guān)基因表達的關(guān)聯(lián)。并進一步通過適應(yīng)性進化和高通量篩選相結(jié)合的方法篩選低產(chǎn)尿素的釀酒酵母菌株,主要結(jié)論如下:(1)釀酒酵母非偏好性氮源尿素的代謝受到氮代謝阻遏效應(yīng)的調(diào)控,而氮代謝阻遏效應(yīng)對釀酒酵母氮源次序利用的調(diào)控主要依賴于4個轉(zhuǎn)錄因子Gln3p、Gat1p、Dal80p、Gzf3p。在營養(yǎng)豐富的培養(yǎng)基中,發(fā)酵末期尿素的積累受到Gln3p和Gat1p正調(diào)控,Dal80p和Gzf3p的負調(diào)控。敲除GZF3基因可使發(fā)酵末期尿素生成量降低18.7%。過量表達GLN3和GAT1基因不能有效降低發(fā)酵末期的尿素生成,而過量表達DAL80和GZF3基因則會增加發(fā)酵末期尿素的生成。過量表達截斷的GLN3和GAT1基因,能有效降低發(fā)酵末期尿素生成,但其對尿素代謝增強的效果依賴GLN3、GAT1和DAL80基因功能的完整性。GZF3基因敲除則能進一步增強過量表達截斷GLN3和GAT1基因?qū)Πl(fā)酵末期尿素含量的降低效果。(2)釀酒酵母中尿素來源于精氨酸的直接降解,而精氨酸轉(zhuǎn)運蛋白在調(diào)控精氨酸利用和尿素生成過程中發(fā)揮重要的作用。通過組合敲除3種精氨酸轉(zhuǎn)運蛋白基因ALP1、GAP1、CAN1發(fā)現(xiàn),在精氨酸為氮源條件下,CAN1基因敲除對精氨酸利用的抑制最嚴重,GAP1基因敲除次之,而ALP1基因敲除無明顯影響。在同等生長條件下,過量表達ALP1基因比CAN1基因更有利于釀酒酵母對精氨酸的利用。三種精氨酸轉(zhuǎn)運蛋白基因?qū)ψ陨砘虮磉_和精氨酸代謝相關(guān)基因的表達可能存在協(xié)同調(diào)控機制。精氨酸轉(zhuǎn)運蛋白基因的組合敲除顯示,相對于野生型菌株,雙敲除GAP1和CAN1基因后,釀酒酵母發(fā)酵末期的尿素含量下降了68.4%。(3)釀酒酵母尿素轉(zhuǎn)運蛋白Dur3p在尿素轉(zhuǎn)運和代謝過程中扮演著重要的角色。通過對尿素轉(zhuǎn)運蛋白Dur3p泛素化位點的預(yù)測,發(fā)現(xiàn)其第556位和第571位賴氨酸是可信度較高的泛素化位點,在此基礎(chǔ)上,對這些泛素化位點進行組合突變。在YNB添加尿素培養(yǎng)基中加入偏好性氮源谷氨酰胺后,Dur3p的泛素化水平提升了95.8%。在YNB添加尿素培養(yǎng)條件下,相對于Dur3p,Dur3p571R的泛素化水平下降14.3%。在YNB培養(yǎng)基中同時添加尿素和谷氨酰胺,相對于Dur3p,Dur3pK556R突變的泛素化水平下降了16.2%。相對于Dur3p,過量表達Dur3p泛素化位點突變體對釀酒酵母的生長沒有顯著的影響,但是能顯著降低發(fā)酵末期尿素的含量。在Δdur3菌株中,相比于Dur3p,過量表達Dur3pK556R和Dur3pK571R突變體后,發(fā)酵末期尿素含量分別下降75.8%和28.3%。(4)釀酒酵母核小體排布與基因的轉(zhuǎn)錄調(diào)控密切相關(guān)。通過高通量測序技術(shù)分析在不同氮源條件下,釀酒酵母基因組上整體的核小體排布,發(fā)現(xiàn)不同氮源條件影響整體的核小體排布和氮代謝阻遏(NCR)相關(guān)基因的表達。此外TATA-containing基因的核小體排布豐度明顯高于TATA-less基因。在高核小體和低核小體排布情況下,相對于谷氨酰胺條件下,由谷氨酰胺培養(yǎng)條件下轉(zhuǎn)移到脯胺酸培養(yǎng)條件后,TATA-less基因編碼區(qū)域的核小體排布豐度有顯著的升高。核小體預(yù)測激活的NCR相關(guān)基因,大部分基因的表達水平都上調(diào),而核小體預(yù)測抑制的NCR基因,基因的表達和核小體預(yù)測之間并沒有顯著的相關(guān)性。(5)基于釀酒酵母尿素代謝機制研究,發(fā)現(xiàn)增強酵母的尿素代謝能力有助于降低發(fā)酵末期尿素的積累。應(yīng)用適應(yīng)性進化策略持續(xù)刺激釀酒酵母N85單倍體菌株尿素代謝能力的進化,并結(jié)合高通量篩選獲得一株低產(chǎn)尿素的適應(yīng)性進化突變菌株。和野生菌相比,突變菌株發(fā)酵末期尿素含量下降了16.8%。在此基礎(chǔ)上,對突變菌株進行全基因組測序和比較基因組學(xué)分析,發(fā)現(xiàn)進化菌株存在170個潛在的突變位點,有27個基因的64個氨基酸位點發(fā)生突變。通過進一步的重測序驗證和對這些基因潛在功能的分析,發(fā)現(xiàn)ESL2基因可能和發(fā)酵末期尿素的降低有關(guān)。敲除N85單倍體的ESL2基因后發(fā)現(xiàn),和野生菌菌株相比,Δesl2菌株發(fā)酵末期尿素含量下降了12.8%。
[Abstract]:Ethyl carbamate is a kind of harmful substance widely existed in fermented food. Its existence seriously affects the safety of traditional fermented food in China. Urea is one of the most important precursor substances in the production of ethyl carbamate in yellow wine, reducing the content of urea in yellow wine at the end of the fermentation of yellow wine and reducing the content of ethyl carbamate in yellow wine. The effect of nitrogen metabolism repression factor, arginine transport pathway and urea transporter Dur3p ubiquitination on the production of urea at the end of fermentation of Saccharomyces cerevisiae, the correlation between the whole nucleosome arrangement and nitrogen metabolism repression related gene expression of Saccharomyces cerevisiae were studied in this paper. The main conclusions are as follows: (1) the metabolism of non preference nitrogen source urea in Saccharomyces cerevisiae is regulated by nitrogen metabolism repression, and the regulation of nitrogen metabolism repression effect on nitrogen source sequence of Saccharomyces cerevisiae is mainly dependent on the 4 transcription factors Gln3p, Gat1p, Dal80p, Gzf. 3p. in the nutrient rich medium, the accumulation of urea at the end of fermentation is regulated by Gln3p and Gat1p, and the negative regulation of Dal80p and Gzf3p. Knockout GZF3 gene can reduce the production of urea at the end of fermentation by 18.7%. overexpression of GLN3 and GAT1 genes, which can not effectively reduce the urea production at the end of fermentation, while excessive expression of DAL80 and GZF3 genes will increase. The overexpression of the truncated GLN3 and GAT1 genes can effectively reduce the production of urea at the end of the fermentation, but its effect on the enhancement of urea metabolism is dependent on GLN3. The completeness of.GZF3 gene knockout of GAT1 and DAL80 gene functions can further increase the overexpression of the reduced GLN3 and GAT1 genes to decrease the urea content at the end of fermentation. (2) urea is derived from the direct degradation of arginine in Saccharomyces cerevisiae, and the arginine transporter plays an important role in the regulation of arginine utilization and urea production. By knocking out 3 arginine transporter genes ALP1, GAP1, and CAN1, the CAN1 gene knocks against arginine under the condition of arginine as the nitrogen source The inhibition was the most serious, GAP1 gene knockout, and ALP1 gene knockout had no obvious effect. Under the same growth condition, overexpression of ALP1 gene was more beneficial to the use of Saccharomyces cerevisiae than CAN1 gene. The expression of three arginine transporter genes may have synergistic regulation on the expression of gene expression and arginine metabolite related genes. Mechanism. Combinatorial knockout of the arginine transporter gene showed that the urea content at the end of fermentation of Saccharomyces cerevisiae decreased by 68.4%. (3) after double knockout of GAP1 and CAN1 genes, compared with wild type strains, and the urea transporter Dur3p played an important role in the process of urea transport and metabolism through the urea transporter Dur3p ubiquitin. It is found that the 556th and 571st lysine are more ubiquitous sites with high reliability. On this basis, these ubiquitination sites are combined mutagenesis. After adding a preference nitrogen source glutamine in the YNB adding urea medium, the ubiquitination level of Dur3p is raised by 95.8%. under the condition of YNB adding urea. In Dur3p, the ubiquitination level of Dur3p571R decreased by 14.3%. in the YNB medium simultaneously with urea and glutamine. Compared with Dur3p, the ubiquitination level of Dur3pK556R mutation decreased by 16.2%. relative to Dur3p. Excessive expression of Dur3p ubiquitination site mutant had no significant effect on the growth of Saccharomyces cerevisiae, but it could significantly reduce the end stage of fermentation. The content of urea in the delta dur3 strain, compared to Dur3p, over expression of Dur3pK556R and Dur3pK571R mutants, the urea content at the end of the fermentation decreased by 75.8% and 28.3%. (4), respectively. The nucleosome arrangement of the Saccharomyces cerevisiae was closely related to the gene transcription regulation. The genome of Saccharomyces cerevisiae was analyzed by high throughput sequencing technology. The overall nucleosome arrangement was found to affect the overall nucleosome arrangement and nitrogen metabolism repression (NCR) related genes. In addition, the nucleosome abundances of the TATA-containing gene were significantly higher than that of the TATA-less gene. Under the condition of high nucleosome and low nucleosome, the glutamine was cultured under glutamine. The abundances of the nucleosome arrangement in the TATA-less gene coding region were significantly higher after the culture conditions were transferred to prolyl acid. The nucleosome predicted the activation of the NCR related genes, and the expression level of most of the genes was up, and there was no significant correlation between the nucleosome predictive NCR gene, the gene expression and the nucleosome prediction. (5) based on the study on the mechanism of urea metabolism in Saccharomyces cerevisiae, it was found that the urea metabolism ability of enhanced yeast could help to reduce the accumulation of urea at the end of fermentation. The adaptive evolutionary strategy was used to stimulate the evolution of the urea metabolism ability of the yeast N85 haploid strain, and the adaptive evolutionary mutation of a low yield urea was obtained by high throughput screening. Strain. Compared with the wild bacteria, the end stage urea content of the mutant strain was reduced by 16.8%.. The whole genome sequencing and comparative genomics analysis of the mutant strain were carried out. 170 potential mutation sites were found in the evolutionary strain and 64 amino acid sites of 27 genes were mutated. The analysis of the potential function of these genes shows that the ESL2 gene may be related to the decrease in the end of the fermentation of urea. After knocking out the ESL2 gene of the N85 haploid, it was found that the urea content at the end of fermentation of the strain of delta esl2 decreased by 12.8%. compared with the wild strain.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS261.1

【參考文獻】

相關(guān)期刊論文 前4條

1 張偉平;趙鑫銳;堵國成;鄒慧君;傅建偉;周景文;陳堅;;釀酒酵母氮代謝物阻遏效應(yīng)及其對發(fā)酵食品安全的影響[J];應(yīng)用與環(huán)境生物學(xué)報;2012年05期

2 倪莉;呂旭聰;黃志清;蔡琪琪;梁爽;饒平凡;;黃酒的生理功效及其生理活性物質(zhì)研究進展[J];中國食品學(xué)報;2012年03期

3 周景文;堵國成;陳堅;;發(fā)酵食品有害氨(胺)類代謝物:形成機制和消除策略[J];中國食品學(xué)報;2011年09期

4 吳世嘉;王洪新;;發(fā)酵食品中氨基甲酸乙酯的研究進展[J];化學(xué)與生物工程;2009年09期

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本文編號：2023062