氧化葡糖桿菌山梨醇代謝相關(guān)基因研究
發(fā)布時間：2018-09-04 17:26
【摘要】：目的:目前生產(chǎn)維生素C (Vitamin c,Vc)的主要方法為我國上世紀70年代首創(chuàng)的Vc二步發(fā)酵法。兩步發(fā)酵法需通過兩次發(fā)酵及三種微生物的共同作用,存在發(fā)酵工藝復雜和微生物代謝污染等問題,目前Vc生產(chǎn)工藝發(fā)展的主流方向是將兩步發(fā)酵簡化為一步單菌發(fā)酵。本實驗室在前期工作中,通過在負責醇糖轉(zhuǎn)化的氧化葡糖桿菌中導入產(chǎn)酸基因簇實現(xiàn)了由D-山梨醇到2-酮基-L-古龍酸(2-keto-L-gulonic acid,2-KGA)的一步單菌發(fā)酵,但醇酸轉(zhuǎn)化率較低。本課題對山梨醇相關(guān)基因及代謝途徑進行了研究,嘗試通過增強主路代謝和阻斷旁路代謝兩方面來提高一步菌醇酸轉(zhuǎn)化率。方法:(1)選取了四種常用和一種文獻報道的氧化葡糖桿菌強啟動子,同時通過氧化葡糖桿菌轉(zhuǎn)錄組測序選取了轉(zhuǎn)錄水平最高的三種啟動子,構(gòu)建山梨糖脫氫酶山梨酮脫氫酶表達載體,電擊轉(zhuǎn)化氧化葡糖桿菌比較2-KGA產(chǎn)量。(2)根據(jù)基因組注釋和轉(zhuǎn)錄組差異分析并結(jié)合文獻調(diào)研預測了 12種可能參與山梨醇代謝的基因,包括3種山梨醇脫氫酶(SLDH)、3種山梨糖還原酶(SR)、3種木糖醇脫氫酶(XDH)和3種酮古龍酸還原酶(HADH),通過課題組建立的快速高效同源重組法對這些酶的編碼基因進行一一敲除,分別以山梨醇、山梨糖為碳源對敲除菌進行生長及糖酸代謝水平評價。選擇表型變化大的敲除菌進行基因回補。部分敲除菌導入山梨糖脫氫酶山梨酮脫氫酶表達載體,比較基因敲除前后菌株醇糖代謝及2-KGA產(chǎn)量的變化。同時,為建立基因無痕敲除操作系統(tǒng)以實現(xiàn)多個相關(guān)基因的敲除,對upp基因進行敲除及回補工作,通過Tn5轉(zhuǎn)座突變篩選其他未知的與旁路代謝相關(guān)基因。結(jié)果:(1)在試管及搖瓶中培養(yǎng),PcsbD啟動子與天然啟動子相比產(chǎn)酸量分別提高15%和19%。(2)目前已分別在氧化葡糖桿菌H24和1.637中成功敲除了 10個和11個基因。分別以山梨醇、山梨糖為碳源對敲除菌進行評價發(fā)現(xiàn),敲除菌H24Asr3以山梨糖為碳源時前期生長緩慢,而敲除菌1.637△sr3在兩種碳源中前期生長緩慢;H24和1.637的srl、sr2、sr3敲除菌以山梨醇為碳源時山梨糖的積累增多,其中1.637△sr3敲除菌山梨糖積累最多,1.637△sr3的回補實驗結(jié)果進一步證明sr3基因影響菌體生長及山梨糖利用。導入山梨糖脫氫酶山梨酮脫氫酶表達載體后,1.637△sr3產(chǎn)酸量高于野生菌1.637。(3)完成了氧化葡糖桿菌upp基因敲除及回補,構(gòu)建pBBR1MCS5-sndhsdh/1.637重組菌株Tn5突變體庫,篩選出20株疑似產(chǎn)酸增強突變株。結(jié)論:(1)篩選得到1個產(chǎn)酸水平高于天然啟動子的啟動子PcsbD,能夠提高醇酸轉(zhuǎn)化率,增強主路代謝。(2)通過對敲除菌評價分析發(fā)現(xiàn)在氧化葡糖桿菌基因組中sr3基因與菌體生長及山梨糖利用相關(guān)。(3)構(gòu)建了氧化葡糖桿菌upp基因敲除菌,回補實驗證明upp能夠作為負篩選標記基因。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q933
本文編號：2222849
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q933
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