锌离子对丙酮丁醇梭菌发酵的调控作用及机制
发布时间：2018-08-05 11:44
【摘要】：生物丁醇发酵工艺存在多方面限制因素,锌(Zn~(2+))作为微生物生命代谢必需微量营养元素及关键辅因子同时受到广泛关注,因而揭示Zn~(2+)在丁醇生产菌株丙酮丁醇梭菌生理代谢的调控作用及机制对挖掘功能基因及建立菌株改造策略等具有重要的理论研究意义。本论文研究工作通过生物过程工程策略、胞内代谢水平分析、转录组学分析及基因工程改造策略等,系统阐释锌离子对丙酮丁醇梭菌丁醇发酵的多效调控作用及机制。外源添加ZnSO4C·7H2O对丙酮丁醇梭菌发酵具有多效调控作用,并确定锌离子最适添加水平为1mg/LZnSO4·7H2O,微量锌离子一方面增强菌体对数生长、碳源利用、有机酸重吸收及溶剂代谢,丁醇产率由0.14g/L/h提高至0.32g/L/h;另一方面增强菌体对甲酸、乙酸、丁酸及丁醇的胁迫耐受性,有效缓解代谢迟滞,其中丁醇耐受水平由15 g/L提高至18 g/L。同时,锌离子与碳酸钙对木糖代谢及丁醇耐受性存在协同调控。对胞内关键代谢物进行LC-ESI/MS分析发现,锌离子调控作用在代谢水平具有全局调控特性。在锌离子响应状态下,发酵前期胞内糖酵解中间代谢产物G6P、F6P、FBP与PEP以及产酸/产溶剂代谢节点产物AcCoA、AcAcCoA与BuCoA含量降低,中心碳代谢转化加快;能量ATP及还原力NADH含量水平提升,促使乙醇/丁醇代谢通路提前开启以维持胞内氧化还原平衡,进而调节中心碳流在关键代谢节点重分配。全局转录组学分析表明锌离子对胞内中心碳代谢、能量代谢及氧化还原平衡等关键基因同样具有全局调控特性。在锌离子响应状态下,多种碳源(二糖、己糖及戊糖)转运与代谢关键基因如葡萄糖转运基因CAC0570(g/cG,12.25倍)、CAC1353与CAC1354、果糖操纵子fru及木糖/阿拉伯糖转运与代谢等相关14个基因转录水平显著上调,糖酵解及产酸/产溶剂代谢相关13个基因转录水平也不同程度上调,但产氢代谢关键基因hydA、转录调控基因abrB,ctsR与hrcA以及胁迫应答基因转录水平下调。基于葡萄糖转运调控策略,在丙酮丁醇梭菌中分别过表达glcG、CAC1353及CAC1354,在基因水平验证锌离子调控机制。过表达CAC1353或CAC1354重组菌株生长代谢出现迟滞;过表达glcG重组菌株ABE发酵性能显著提高,发酵周期缩短至28 h,丁醇产量及产率最高可达13.9 g/L及0.50 g/L/h,且甲酸、乙酸、丁酸及丁醇胁迫耐受性增强,其中在7.5 g/L乙酸胁迫条件下,丁醇产量可达16.4 g/L。LC-ESI/MS分析表明过表达glcG同样具有全局调控特性,引发中心碳流通量与辅因子(ATP与NADH)含量水平的级联调控,证实锌离子通过提高葡萄糖转运基因glcG表达水平而对胞内代谢进行全局调控。以预处理玉米秸秆酶解液为碳源的ABE发酵,丁醇产率由0.18g/L/h大幅提高至0.44 g/L/h,因而该调控策略可提高木质纤维素类生物质发酵生产丁醇的竞争力。
[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.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ923
本文编号：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.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ923
【参考文献】
相关期刊论文 前3条
1 徐桂红;赵心清;李宁;白凤武;;锌离子提高絮凝酵母乙酸胁迫耐受性[J];化工学报;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 陈丽杰;辛程勋;邓攀;任剑刚;梁环环;白凤武;;丙酮丁醇梭菌发酵菊芋汁生产丁醇[J];生物工程学报;2010年07期
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