氧化葡糖杆菌山梨醇代谢相关基因研究

发布时间：2018-09-04 17:26

【摘要】：目的:目前生产维生素C (Vitamin c,Vc)的主要方法为我国上世纪70年代首创的Vc二步发酵法。两步发酵法需通过两次发酵及三种微生物的共同作用,存在发酵工艺复杂和微生物代谢污染等问题,目前Vc生产工艺发展的主流方向是将两步发酵简化为一步单菌发酵。本实验室在前期工作中,通过在负责醇糖转化的氧化葡糖杆菌中导入产酸基因簇实现了由D-山梨醇到2-酮基-L-古龙酸(2-keto-L-gulonic acid,2-KGA)的一步单菌发酵,但醇酸转化率较低。本课题对山梨醇相关基因及代谢途径进行了研究,尝试通过增强主路代谢和阻断旁路代谢两方面来提高一步菌醇酸转化率。方法:(1)选取了四种常用和一种文献报道的氧化葡糖杆菌强启动子,同时通过氧化葡糖杆菌转录组测序选取了转录水平最高的三种启动子,构建山梨糖脱氢酶山梨酮脱氢酶表达载体,电击转化氧化葡糖杆菌比较2-KGA产量。(2)根据基因组注释和转录组差异分析并结合文献调研预测了 12种可能参与山梨醇代谢的基因,包括3种山梨醇脱氢酶(SLDH)、3种山梨糖还原酶(SR)、3种木糖醇脱氢酶(XDH)和3种酮古龙酸还原酶(HADH),通过课题组建立的快速高效同源重组法对这些酶的编码基因进行一一敲除,分别以山梨醇、山梨糖为碳源对敲除菌进行生长及糖酸代谢水平评价。选择表型变化大的敲除菌进行基因回补。部分敲除菌导入山梨糖脱氢酶山梨酮脱氢酶表达载体,比较基因敲除前后菌株醇糖代谢及2-KGA产量的变化。同时,为建立基因无痕敲除操作系统以实现多个相关基因的敲除,对upp基因进行敲除及回补工作,通过Tn5转座突变筛选其他未知的与旁路代谢相关基因。结果:(1)在试管及摇瓶中培养,PcsbD启动子与天然启动子相比产酸量分别提高15%和19%。(2)目前已分别在氧化葡糖杆菌H24和1.637中成功敲除了 10个和11个基因。分别以山梨醇、山梨糖为碳源对敲除菌进行评价发现,敲除菌H24Asr3以山梨糖为碳源时前期生长缓慢,而敲除菌1.637△sr3在两种碳源中前期生长缓慢;H24和1.637的srl、sr2、sr3敲除菌以山梨醇为碳源时山梨糖的积累增多,其中1.637△sr3敲除菌山梨糖积累最多,1.637△sr3的回补实验结果进一步证明sr3基因影响菌体生长及山梨糖利用。导入山梨糖脱氢酶山梨酮脱氢酶表达载体后,1.637△sr3产酸量高于野生菌1.637。(3)完成了氧化葡糖杆菌upp基因敲除及回补,构建pBBR1MCS5-sndhsdh/1.637重组菌株Tn5突变体库,筛选出20株疑似产酸增强突变株。结论:(1)筛选得到1个产酸水平高于天然启动子的启动子PcsbD,能够提高醇酸转化率,增强主路代谢。(2)通过对敲除菌评价分析发现在氧化葡糖杆菌基因组中sr3基因与菌体生长及山梨糖利用相关。(3)构建了氧化葡糖杆菌upp基因敲除菌,回补实验证明upp能够作为负筛选标记基因。
[Abstract]:Objective: at present, the main method of producing vitamin C (Vitamin C) is Vc two-step fermentation method, which was first developed in China in 1970s. Two-step fermentation requires two fermentation and the interaction of three microbes, there are some problems such as complex fermentation process and microbial metabolic pollution. At present, the main direction of Vc production process is to simplify the two-step fermentation to one-step fermentation. In our previous work, the one-step fermentation from D-sorbitol to 2-keto-L-gulonic acid (2-keto-L-gulonic acid,2-KGA) was achieved by introducing the acid-producing gene cluster into glucose-oxidized bacilli responsible for glycosyl conversion, but the conversion rate of alkyd was relatively low. In this study, sorbitol related genes and metabolic pathways were studied to improve the conversion rate of alkyd by enhancing the metabolism of the main pathway and blocking the metabolism of the bypass pathway. Methods: (1) four common and one reported strong promoter of Glucobacter oxide were selected, and the three promoters with the highest transcriptional level were selected by sequencing the transcriptome of Glucobacter oxide. The expression vector of sorbitone dehydrogenase was constructed, and the electroporation of glucose-oxidized bacillus to compare the 2-KGA yield. (2) according to genome annotation and transcriptional differential analysis, 12 genes that might be involved in sorbitol metabolism were predicted. Including three sorbitol dehydrogenase (SLDH), three sorbitan reductase (SR), three xylitol dehydrogenase (XDH) and three ketocolonic acid reductase (HADH), the coding genes of these enzymes were knocked out one by a rapid and efficient homologous recombination method established by our team. Sorbitol and sorbose were used as carbon source to evaluate the growth and metabolism of saccharic acid. Selection of knockout bacteria with large phenotypic changes for gene recovery. Some knockout bacteria were introduced into sorbitone dehydrogenase expression vector to compare the changes of alcohol glucose metabolism and 2-KGA yield before and after gene knockout. At the same time, in order to set up a non-trace knockout operating system to realize the knockout of many related genes, the upp gene was knocked out and compensated. Other unknown genes related to bypass metabolism were screened by Tn5 transposition mutation. Results: (1) the acid production of PcsbD promoter cultured in test tube and shaking flask was increased by 15% and 19%, respectively, compared with the natural promoter. (2) 10 and 11 genes had been successfully knocked out in Gluconobacter oxidans H24 and 1.637, respectively. Using sorbitol and sorbitum as carbon source, the results showed that the growth of knockout H24Asr3 was slow in the early stage when sorbitol was used as carbon source. However, the accumulation of sorbitum increased in the srl,sr2,sr3 knockout of 1.637 sr3 with sorbitol as carbon source, which grew slowly in the early stage of the two carbon sources. The results of 1.637 sr3 knockout experiment showed that sr3 gene affected the growth of bacteria and the utilization of sorbitose. The result of 1.637 sr3 knockout experiment showed that sorbitum accumulated the most amount of sorbitum at 1.637 sr3. After the introduction of sorbitone dehydrogenase expression vector, the acid production at 1.637 sr3 was higher than that of wild strain 1.637. (3) the upp gene knockout and complement of Glucobacter oxidans were completed, and the Tn5 mutants library of pBBR1MCS5-sndhsdh/1.637 recombinant strain was constructed, and 20 suspected acid-producing enhanced mutants were screened out. Conclusion: (1) screening a promoter with a higher level of acid production than that of a natural promoter can improve the conversion rate of alkyd. (2) it was found that sr3 gene was related to cell growth and sorbitose utilization in the genome of Glucobacter oxidans by the evaluation of knockout bacteria. (3) the upp gene knockout bacteria of Glucobacter oxidans were constructed. The compensation experiment showed that upp can be used as a negative screening marker gene.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q933

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