鋅離子對丙酮丁醇梭菌發(fā)酵的調(diào)控作用及機制
發(fā)布時間：2018-08-05 11:44
【摘要】：生物丁醇發(fā)酵工藝存在多方面限制因素,鋅(Zn~(2+))作為微生物生命代謝必需微量營養(yǎng)元素及關(guān)鍵輔因子同時受到廣泛關(guān)注,因而揭示Zn~(2+)在丁醇生產(chǎn)菌株丙酮丁醇梭菌生理代謝的調(diào)控作用及機制對挖掘功能基因及建立菌株改造策略等具有重要的理論研究意義。本論文研究工作通過生物過程工程策略、胞內(nèi)代謝水平分析、轉(zhuǎn)錄組學(xué)分析及基因工程改造策略等,系統(tǒng)闡釋鋅離子對丙酮丁醇梭菌丁醇發(fā)酵的多效調(diào)控作用及機制。外源添加ZnSO4C·7H2O對丙酮丁醇梭菌發(fā)酵具有多效調(diào)控作用,并確定鋅離子最適添加水平為1mg/LZnSO4·7H2O,微量鋅離子一方面增強菌體對數(shù)生長、碳源利用、有機酸重吸收及溶劑代謝,丁醇產(chǎn)率由0.14g/L/h提高至0.32g/L/h;另一方面增強菌體對甲酸、乙酸、丁酸及丁醇的脅迫耐受性,有效緩解代謝遲滯,其中丁醇耐受水平由15 g/L提高至18 g/L。同時,鋅離子與碳酸鈣對木糖代謝及丁醇耐受性存在協(xié)同調(diào)控。對胞內(nèi)關(guān)鍵代謝物進行LC-ESI/MS分析發(fā)現(xiàn),鋅離子調(diào)控作用在代謝水平具有全局調(diào)控特性。在鋅離子響應(yīng)狀態(tài)下,發(fā)酵前期胞內(nèi)糖酵解中間代謝產(chǎn)物G6P、F6P、FBP與PEP以及產(chǎn)酸/產(chǎn)溶劑代謝節(jié)點產(chǎn)物AcCoA、AcAcCoA與BuCoA含量降低,中心碳代謝轉(zhuǎn)化加快;能量ATP及還原力NADH含量水平提升,促使乙醇/丁醇代謝通路提前開啟以維持胞內(nèi)氧化還原平衡,進而調(diào)節(jié)中心碳流在關(guān)鍵代謝節(jié)點重分配。全局轉(zhuǎn)錄組學(xué)分析表明鋅離子對胞內(nèi)中心碳代謝、能量代謝及氧化還原平衡等關(guān)鍵基因同樣具有全局調(diào)控特性。在鋅離子響應(yīng)狀態(tài)下,多種碳源(二糖、己糖及戊糖)轉(zhuǎn)運與代謝關(guān)鍵基因如葡萄糖轉(zhuǎn)運基因CAC0570(g/cG,12.25倍)、CAC1353與CAC1354、果糖操縱子fru及木糖/阿拉伯糖轉(zhuǎn)運與代謝等相關(guān)14個基因轉(zhuǎn)錄水平顯著上調(diào),糖酵解及產(chǎn)酸/產(chǎn)溶劑代謝相關(guān)13個基因轉(zhuǎn)錄水平也不同程度上調(diào),但產(chǎn)氫代謝關(guān)鍵基因hydA、轉(zhuǎn)錄調(diào)控基因abrB,ctsR與hrcA以及脅迫應(yīng)答基因轉(zhuǎn)錄水平下調(diào)。基于葡萄糖轉(zhuǎn)運調(diào)控策略,在丙酮丁醇梭菌中分別過表達glcG、CAC1353及CAC1354,在基因水平驗證鋅離子調(diào)控機制。過表達CAC1353或CAC1354重組菌株生長代謝出現(xiàn)遲滯;過表達glcG重組菌株ABE發(fā)酵性能顯著提高,發(fā)酵周期縮短至28 h,丁醇產(chǎn)量及產(chǎn)率最高可達13.9 g/L及0.50 g/L/h,且甲酸、乙酸、丁酸及丁醇脅迫耐受性增強,其中在7.5 g/L乙酸脅迫條件下,丁醇產(chǎn)量可達16.4 g/L。LC-ESI/MS分析表明過表達glcG同樣具有全局調(diào)控特性,引發(fā)中心碳流通量與輔因子(ATP與NADH)含量水平的級聯(lián)調(diào)控,證實鋅離子通過提高葡萄糖轉(zhuǎn)運基因glcG表達水平而對胞內(nèi)代謝進行全局調(diào)控。以預(yù)處理玉米秸稈酶解液為碳源的ABE發(fā)酵,丁醇產(chǎn)率由0.18g/L/h大幅提高至0.44 g/L/h,因而該調(diào)控策略可提高木質(zhì)纖維素類生物質(zhì)發(fā)酵生產(chǎn)丁醇的競爭力。
[Abstract]:There are many limiting factors in biological butanol fermentation process. Zinc (Zn ~ (2), as a necessary micronutrient element and key cofactor of microbial life metabolism, has been paid more and more attention at the same time. Therefore, it is of great theoretical significance to reveal the regulation and mechanism of Zn ~ (2) in the physiological metabolism of Clostridium acetone, a butanol producing strain, for excavating functional genes and establishing the transformation strategy of the strain. Through biological process engineering strategy, intracellular metabolic level analysis, transcriptome analysis and genetic engineering strategy, this paper systematically explained the effect and mechanism of zinc ion on butanol fermentation of Clostridium acetone. Exogenous ZnSO4C 7H2O could regulate the fermentation of Clostridium acetone butanol, and the optimum level of zinc ion was 1mg/LZnSO4 _ 7H _ 2O. On the one hand, trace zinc ion enhanced cell logarithm growth, carbon source utilization, organic acid reabsorption and solvent metabolism. The yield of butanol was increased from 0.14g/L/h to 0.32g / L / h, on the other hand, the stress tolerance to formic acid, acetic acid, butyric acid and butanol was enhanced, and the metabolic retardation was effectively alleviated, in which the level of butanol tolerance increased from 15 g / L to 18 g / L. At the same time, zinc ion and calcium carbonate have synergistic regulation on xylose metabolism and butanol tolerance. LC-ESI/MS analysis of the key metabolites in the cell showed that the zinc ion regulation had global regulatory characteristics at the metabolic level. Under the zinc ion response, the contents of G6PnF6PnFBP and PEP and accoAn / solvent-producing metabolite AcCoA and BuCoA decreased, the transformation of central carbon was accelerated, and the content of energy ATP and reductive power NADH increased in the early stage of fermentation. The ethanol / butanol metabolic pathway was opened in advance to maintain redox balance and regulate the redistribution of central carbon flow at key metabolic nodes. Global transcriptome analysis showed that zinc ions also had global regulation on the central carbon metabolism, energy metabolism and redox balance. In a zinc ion-responsive state, a variety of carbon sources (disaccharides, The transcription levels of 14 genes related to hexose and pentose transport and metabolism, such as glucose transport gene CAC0570 (g / cGN 12.25-fold), fructose operon fru and xylose / arabinose transport and metabolism, were significantly up-regulated. The transcription levels of 13 genes related to glycolysis and acid / solvent metabolism were up-regulated in varying degrees, but the transcription levels of the key genes of hydrogen production, hydA, abrBCtsR and hrcA, and stress response genes were down-regulated. Based on the glucose transport regulation strategy, glcGfCAC1353 and CAC1354 were overexpressed in Clostridium acetone butanol, respectively. The mechanism of zinc ion regulation was verified at the gene level. The growth and metabolism of over-expressed CAC1353 or CAC1354 recombinant strains were delayed, the fermentation performance of over-expressed glcG recombinant strain ABE was significantly improved, the fermentation period was shortened to 28 h, the highest yield and yield of butanol were 13.9 g / L and 0.50 g / L / h, and formic acid and acetic acid, The tolerance of butyric acid and butanol stress was enhanced. Under the condition of 7.5 g / L acetic acid stress, the yield of butanol reached 16.4 g/L.LC-ESI/MS. The cascade regulation of central carbon fluxes and cofactors (ATP and NADH) levels confirmed that zinc ions regulate intracellular metabolism globally by increasing the expression level of glucose transporter gene glcG. The yield of butanol was significantly increased from 0.18g/L/h to 0.44 g / L / h by ABE fermentation with pretreatment of corn straw enzymatic hydrolysis solution, so the control strategy could improve the competitiveness of lignocellulosic biomass fermentation to produce butanol.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ923
本文編號：2165695
[Abstract]:There are many limiting factors in biological butanol fermentation process. Zinc (Zn ~ (2), as a necessary micronutrient element and key cofactor of microbial life metabolism, has been paid more and more attention at the same time. Therefore, it is of great theoretical significance to reveal the regulation and mechanism of Zn ~ (2) in the physiological metabolism of Clostridium acetone, a butanol producing strain, for excavating functional genes and establishing the transformation strategy of the strain. Through biological process engineering strategy, intracellular metabolic level analysis, transcriptome analysis and genetic engineering strategy, this paper systematically explained the effect and mechanism of zinc ion on butanol fermentation of Clostridium acetone. Exogenous ZnSO4C 7H2O could regulate the fermentation of Clostridium acetone butanol, and the optimum level of zinc ion was 1mg/LZnSO4 _ 7H _ 2O. On the one hand, trace zinc ion enhanced cell logarithm growth, carbon source utilization, organic acid reabsorption and solvent metabolism. The yield of butanol was increased from 0.14g/L/h to 0.32g / L / h, on the other hand, the stress tolerance to formic acid, acetic acid, butyric acid and butanol was enhanced, and the metabolic retardation was effectively alleviated, in which the level of butanol tolerance increased from 15 g / L to 18 g / L. At the same time, zinc ion and calcium carbonate have synergistic regulation on xylose metabolism and butanol tolerance. LC-ESI/MS analysis of the key metabolites in the cell showed that the zinc ion regulation had global regulatory characteristics at the metabolic level. Under the zinc ion response, the contents of G6PnF6PnFBP and PEP and accoAn / solvent-producing metabolite AcCoA and BuCoA decreased, the transformation of central carbon was accelerated, and the content of energy ATP and reductive power NADH increased in the early stage of fermentation. The ethanol / butanol metabolic pathway was opened in advance to maintain redox balance and regulate the redistribution of central carbon flow at key metabolic nodes. Global transcriptome analysis showed that zinc ions also had global regulation on the central carbon metabolism, energy metabolism and redox balance. In a zinc ion-responsive state, a variety of carbon sources (disaccharides, The transcription levels of 14 genes related to hexose and pentose transport and metabolism, such as glucose transport gene CAC0570 (g / cGN 12.25-fold), fructose operon fru and xylose / arabinose transport and metabolism, were significantly up-regulated. The transcription levels of 13 genes related to glycolysis and acid / solvent metabolism were up-regulated in varying degrees, but the transcription levels of the key genes of hydrogen production, hydA, abrBCtsR and hrcA, and stress response genes were down-regulated. Based on the glucose transport regulation strategy, glcGfCAC1353 and CAC1354 were overexpressed in Clostridium acetone butanol, respectively. The mechanism of zinc ion regulation was verified at the gene level. The growth and metabolism of over-expressed CAC1353 or CAC1354 recombinant strains were delayed, the fermentation performance of over-expressed glcG recombinant strain ABE was significantly improved, the fermentation period was shortened to 28 h, the highest yield and yield of butanol were 13.9 g / L and 0.50 g / L / h, and formic acid and acetic acid, The tolerance of butyric acid and butanol stress was enhanced. Under the condition of 7.5 g / L acetic acid stress, the yield of butanol reached 16.4 g/L.LC-ESI/MS. The cascade regulation of central carbon fluxes and cofactors (ATP and NADH) levels confirmed that zinc ions regulate intracellular metabolism globally by increasing the expression level of glucose transporter gene glcG. The yield of butanol was significantly increased from 0.18g/L/h to 0.44 g / L / h by ABE fermentation with pretreatment of corn straw enzymatic hydrolysis solution, so the control strategy could improve the competitiveness of lignocellulosic biomass fermentation to produce butanol.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ923
【參考文獻】
相關(guān)期刊論文 前3條
1 徐桂紅;趙心清;李寧;白鳳武;;鋅離子提高絮凝酵母乙酸脅迫耐受性[J];化工學(xué)報;2012年06期
2 ;Optimization of butanol production from corn straw hydrolysate by Clostridium acetobutylicum using response surface method[J];Chinese Science Bulletin;2011年14期
3 陳麗杰;辛程勛;鄧攀;任劍剛;梁環(huán)環(huán);白鳳武;;丙酮丁醇梭菌發(fā)酵菊芋汁生產(chǎn)丁醇[J];生物工程學(xué)報;2010年07期
，本文編號：2165695
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