Corynebacterium glutamicum血红素转运蛋白缺失对血红素代谢调控的影响

发布时间：2018-05-19 17:56

本文选题：谷氨酸棒杆菌 + 血红素转运蛋白　；参考：《山东大学》2017年硕士论文

【摘要】：血红素是普遍存在于生物体内的一种生物大分子,它可以作为辅基参与生物的多种生命代谢活动,同时,还可以为生物体提供铁元素。但过量的游离血红素具有毒害作用,谷氨酸棒杆菌中存在精致且复杂的调控系统来确保生物体内血红素的动态平衡。我们在利用谷氨酸棒杆菌生产ALA时,菌体积累过量下游产物,致使菌体的生长乃至呼吸代谢均受到影响。鉴于血红素的重要生理功能,在利用重组菌生产ALA时,血红素合成途径不能被阻断或敲除,只能调控弱化。谷氨酸棒杆菌中的血红素受到两个双组份系统HrrSA和ChrSA的调控作用,主要在转录水平上进行调控。HrrSA主要用来调控血红素合成途径基因转录水平,以及血红素代谢途径相关基因转录水平。ChrSA只有胞内游离血红素的浓度超过菌体耐受性时才转录表达,激活血红素外运蛋白HrtBA基因表达,将胞内过量的游离血红素转运到胞外。本论文首先构建了谷氨酸棒杆菌血红素外运蛋白基因单敲菌株WT-H,血红素外运蛋白与内运蛋白基因同时缺失菌株WT-HH。研究血红素在胞内积累时,对血红素合成途径以及代谢调控途径的影响。结果表明,菌体血红素转运蛋白缺失,胞内游离血红素的量维持动态平衡。菌株积累大量的血红素代谢中间产物,并分泌到胞外。负责调节胞内游离血红素动态平衡的两个双组份系统HrrSA与ChrSA的转录水平上调,血红素合成途径基因转录水平下调,血红素氧化酶基因转录水平与血红素结合蛋白基因转录水平均上调。这是由于游离血红素在胞内积累,菌体通过双组份系统调控胞内游离血红素的动态平衡。我们在谷氨酸棒杆菌hemB基因C端添加降解标签,可有效降低胞内ALA脱水酶的活性,菌体积累的胆色素原明显降低,卟啉化合物的积累量也降低。敲除血红素转运蛋白基因,菌体胞内游离血红素的量仍可以保持动态平衡。其次我们在野生型谷氨酸棒杆菌与血红素转运蛋白基因缺失菌中过表达ALA合成基因,研究过量合成ALA时,对血红素代谢调控的影响。ALA过量积累时,胞内游离血红素的量维持动态平衡;血红素转运蛋白的缺失有利于提高ALA的积累。此时,菌液颜色加深,发酵液中积累大量的卟啉化合物。双组份基因转录水平上调,血红素合成途径基因转录水平也上调。这是由于胞内过量的ALA促使下游代谢提高,积累过量的中间代谢产物。同时菌体双组份系统调节胞内血红素动态平衡。在hemB基因末端添加降解标签可有效提高ALA的积累。综上所述,血红素转运蛋白缺失,胞内游离血红素的量维持动态平衡,菌体积累大量的卟啉化合物。菌株主要通过双组份系统在转录水平调控胞内游离血红素的量,抑制血红素合成途径基因转录水平,提高血红素代谢相关基因转录水平。降低ALA下游代谢产物的积累可有效提高ALA的产量。研究谷氨酸棒杆菌血红素转运蛋白缺失对血红素代谢调控的影响,可以为后期研究血红素以及ALA提供理论依据。
[Abstract]:Heme is a kind of biological macromolecule, which is ubiquitous in organisms. It can be used as an auxiliary group to participate in many biological metabolism activities of organisms, and also provide iron elements for organisms. But excessive free heme has toxic effects. There is a sophisticated and complex regulatory system in Corynebacterium glutaminus to ensure blood red in living organisms. During the production of ALA with Corynebacterium glutamis, the bacteria accumulated over the downstream products, causing the growth of the bacteria and even the respiratory metabolites to be affected. In view of the important physiological function of heme, the heme synthesis pathway can not be blocked or knocked out when the recombinant bacteria are used to produce ALA. The glutamic rod can only be regulated and weakened. The heme of the bacteria is regulated by two bicomponent systems HrrSA and ChrSA. The regulation of.HrrSA mainly at the transcriptional level is mainly used to regulate the transcriptional level of the heme synthesis pathway gene, as well as the transcription level of the heme metabolic pathway related gene.ChrSA only when the concentration of intracellular free heme exceeds the bacterial tolerance. The expression of heme exoprotein HrtBA activates the expression of the heme exoprotein gene and transtransport the intracellular free heme to the extracellular. Firstly, the heme exoprotein gene WT-H, heme exoprotein and the endosin gene are simultaneously missing from the heme strain WT-HH. to study the heme accumulation of heme in the cell. The results showed that the heme transporter was missing and the amount of intracellular free heme maintained dynamic balance. The strain accumulated a large number of intermediate products of heme metabolism and secreted to the extracellular. The transfer of the two components of the intracellular free heme, HrrSA and ChrSA, was responsible for regulating the dynamic balance of intracellular free heme. The transcriptional level of heme synthesis pathway is down regulated, the transcription level of heme oxidase gene and the transcription level of heme binding protein gene are all up-regulated. This is due to the accumulation of heme in the cell and the dynamic balance of intracellular free heme by the bicomponent system. We are in the hemB base of Corynebacterium glutamias. The activity of intracellular ALA dehydrase was effectively reduced because of the addition of degradation labels at the C end. The accumulation of bacterial pigments in the mycelium was significantly reduced and the accumulation of porphyrin compounds decreased. The amount of intracellular free heme in the cell could remain dynamic balance. When the ALA gene was overexpressed in the protein gene deletion bacteria, the amount of intracellular free heme maintained dynamic balance when excessive accumulation of heme metabolism, and the loss of heme transporter was beneficial to the accumulation of ALA when excessive accumulation of ALA in the heme metabolism regulation. The color of the heme transporter was beneficial to the accumulation of ALA. At this time, the color of the bacteria was deepened and the accumulation of porphyrin in the fermented liquid was great. The transcriptional level of the bicomponent gene was up-regulated and the transcriptional level of the heme synthesis pathway was also up-regulated. This is due to the excessive intracellular ALA that promotes downstream metabolism and accumulates excessive intermediate metabolites. At the same time, the bicomponent system regulates the dynamic balance of intracellular heme. The addition of degradation labels at the end of the hemB gene can effectively improve the product of ALA. To sum up, the heme transporter is missing, the amount of intracellular free heme maintains dynamic balance, and the bacteria accumulate a large number of porphyrin compounds. The strain mainly regulates the amount of intracellular free heme at the transcriptional level, inhibits the transcription level of heme synthesis pathway gene, and improves the transcription water of heme metabolism related genes. Lowering the accumulation of metabolites in the downstream ALA can effectively improve the production of ALA. The study of the effect of heme metabolism in heme of Corynebacterium glutamatsu can provide a theoretical basis for the later study of heme and ALA.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51

【相似文献】