酿酒酵母氮代谢阻遏机制解析和低产尿素黄酒发酵菌株选育

发布时间：2018-06-15 18:20

本文选题：氮代谢阻遏 + 精氨酸代谢　；参考：《江南大学》2017年博士论文

【摘要】：氨基甲酸乙酯是一种广泛存在于发酵食品中的有害物质,它的存在严重影响了我国传统发酵食品的安全性。尿素是黄酒中氨基甲酸乙酯生成最重要的前体物质之一,降低发酵末期黄酒中尿素的含量对于黄酒中氨基甲酸乙酯含量的降低具有重要意义。本文通过模式菌株研究氮代谢阻遏调控因子、精氨酸转运途径和尿素转运蛋白Dur3p泛素化对酿酒酵母发酵末期尿素生成的影响,酿酒酵母整体核小体排布与氮代谢阻遏相关基因表达的关联。并进一步通过适应性进化和高通量筛选相结合的方法筛选低产尿素的酿酒酵母菌株,主要结论如下:(1)酿酒酵母非偏好性氮源尿素的代谢受到氮代谢阻遏效应的调控,而氮代谢阻遏效应对酿酒酵母氮源次序利用的调控主要依赖于4个转录因子Gln3p、Gat1p、Dal80p、Gzf3p。在营养丰富的培养基中,发酵末期尿素的积累受到Gln3p和Gat1p正调控,Dal80p和Gzf3p的负调控。敲除GZF3基因可使发酵末期尿素生成量降低18.7%。过量表达GLN3和GAT1基因不能有效降低发酵末期的尿素生成,而过量表达DAL80和GZF3基因则会增加发酵末期尿素的生成。过量表达截断的GLN3和GAT1基因,能有效降低发酵末期尿素生成,但其对尿素代谢增强的效果依赖GLN3、GAT1和DAL80基因功能的完整性。GZF3基因敲除则能进一步增强过量表达截断GLN3和GAT1基因对发酵末期尿素含量的降低效果。(2)酿酒酵母中尿素来源于精氨酸的直接降解,而精氨酸转运蛋白在调控精氨酸利用和尿素生成过程中发挥重要的作用。通过组合敲除3种精氨酸转运蛋白基因ALP1、GAP1、CAN1发现,在精氨酸为氮源条件下,CAN1基因敲除对精氨酸利用的抑制最严重,GAP1基因敲除次之,而ALP1基因敲除无明显影响。在同等生长条件下,过量表达ALP1基因比CAN1基因更有利于酿酒酵母对精氨酸的利用。三种精氨酸转运蛋白基因对自身基因表达和精氨酸代谢相关基因的表达可能存在协同调控机制。精氨酸转运蛋白基因的组合敲除显示,相对于野生型菌株,双敲除GAP1和CAN1基因后,酿酒酵母发酵末期的尿素含量下降了68.4%。(3)酿酒酵母尿素转运蛋白Dur3p在尿素转运和代谢过程中扮演着重要的角色。通过对尿素转运蛋白Dur3p泛素化位点的预测,发现其第556位和第571位赖氨酸是可信度较高的泛素化位点,在此基础上,对这些泛素化位点进行组合突变。在YNB添加尿素培养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添加尿素培养条件下,相对于Dur3p,Dur3p571R的泛素化水平下降14.3%。在YNB培养基中同时添加尿素和谷氨酰胺,相对于Dur3p,Dur3pK556R突变的泛素化水平下降了16.2%。相对于Dur3p,过量表达Dur3p泛素化位点突变体对酿酒酵母的生长没有显著的影响,但是能显著降低发酵末期尿素的含量。在Δdur3菌株中,相比于Dur3p,过量表达Dur3pK556R和Dur3pK571R突变体后,发酵末期尿素含量分别下降75.8%和28.3%。(4)酿酒酵母核小体排布与基因的转录调控密切相关。通过高通量测序技术分析在不同氮源条件下,酿酒酵母基因组上整体的核小体排布,发现不同氮源条件影响整体的核小体排布和氮代谢阻遏(NCR)相关基因的表达。此外TATA-containing基因的核小体排布丰度明显高于TATA-less基因。在高核小体和低核小体排布情况下,相对于谷氨酰胺条件下,由谷氨酰胺培养条件下转移到脯胺酸培养条件后,TATA-less基因编码区域的核小体排布丰度有显著的升高。核小体预测激活的NCR相关基因,大部分基因的表达水平都上调,而核小体预测抑制的NCR基因,基因的表达和核小体预测之间并没有显著的相关性。(5)基于酿酒酵母尿素代谢机制研究,发现增强酵母的尿素代谢能力有助于降低发酵末期尿素的积累。应用适应性进化策略持续刺激酿酒酵母N85单倍体菌株尿素代谢能力的进化,并结合高通量筛选获得一株低产尿素的适应性进化突变菌株。和野生菌相比,突变菌株发酵末期尿素含量下降了16.8%。在此基础上,对突变菌株进行全基因组测序和比较基因组学分析,发现进化菌株存在170个潜在的突变位点,有27个基因的64个氨基酸位点发生突变。通过进一步的重测序验证和对这些基因潜在功能的分析,发现ESL2基因可能和发酵末期尿素的降低有关。敲除N85单倍体的ESL2基因后发现,和野生菌菌株相比,Δesl2菌株发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.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS261.1

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