酚醛抑制物生物脱毒的分子生物学解析及其关键基因元器件库的构建
发布时间：2019-05-30 05:54
【摘要】：酚醛抑制物是木质纤维素生物炼制过程的预处理步骤产生的主要抑制物之一,其主要源于木质素的过度降解,其代表性化合物包括隶属p-羟基苯基类的4-羟基苯甲醛、丁香基类的丁香醛和愈创木酚基类的香草醛。与呋喃类(糠醛和5-羟甲基糠醛)和弱酸类(乙酸、甲酸和乙酰丙酸)抑制物不同,种类较多的酚醛抑制物难挥发且水溶性较差,其芳香环结构致使其降解缓慢。因此,酚醛抑制物是纤维素酶和发酵微生物的主要抑制物。生物脱毒是一种全新的脱毒概念,被认为是未来生物炼制过程不可或缺的步骤,其主要思路是利用具有生物降解能力的微生物转化木质纤维素预处理过程产生的抑制物。目前,对呋喃醛和有机酸生物脱毒机理的研究比较清楚。酚醛抑制物因为水溶性差而难以精确定性定量,因此其生物脱毒机理报道较少。本研究采用RNA-Seq技术和DNA芯片技术,对生物炼制菌株树脂枝孢霉菌(Amorphotheca resinae ZN1)和运动发酵单胞菌(Zymomonas mobilis ZM4)的酚醛抑制物代谢途径和耐受机制进行了解析；对酚醛抑制物生物脱毒关键基因进行了筛选,并在运动发酵单胞菌进行了酚醛抑制物代谢途径的强化改造,初步测试了酚醛抑制物生物脱毒与纤维素乙醇发酵的整合生物加工菌株的能力；主要依据转录组数据,构建了细菌、酵母和霉菌的酚类、呋喃类和弱酸类抑制物生物脱毒的关键基因元器件库。第一部分,为了探究专司生物脱毒的丝状真菌A.resinae ZN1转化酚醛抑制物的机制,本研究通过RNA-Seq技术考察了A. resinae ZN1脱毒酚醛抑制物生成酚酸和酚醇的转录组。结果发现,534个、1576个和1261个基因分别在4-羟基苯甲醛、丁香醛和香草醛转化过程中显著差异表达。GO分析发现,氧化还原和转运是A. resinae ZN1脱毒酚醛抑制物的主要生物学过程。基于推测的A. resinae ZN1转化酚醛抑制物的代谢途径,本研究发现醇脱氢酶、酰基醇脱氢酶和醛还原酶是A. resinae ZN1还原酚醛抑制物产醇代谢途径的关键酶,醛脱氢酶是A. resinae ZN1氧化酚醛抑制物产酸代谢途径的关键酶。第二部分,为了探究产乙醇细菌Z. mobilis ZM4转化酚醛抑制物的机制,本研究通过DNA芯片技术考察了Z. mobilis ZM4还原酚醛抑制物产醇的转录组。结果发现,442个、67个和306个基因分别在4-羟基苯甲醛、丁香醛和香草醛胁迫下显著差异表达。还原、转运和调控是Z. mobilis ZM4还原酚醛抑制物的主要分子机制。本研究鉴定了Zmobilis ZM4还原酚醛抑制物的72个关键基因,其中包括酚醛抑制物胁迫下均显著差异上调表达的ZMO1116 (Oxidoreductase)和ZMO1885 (NADH:flavin oxidoreductase/NADH oxidase)。同时,本研究通过基因组图谱定位发现,在至少2种酚醛抑制物胁迫下显著差异上调表达的272个基因涉及36个基因簇,560个显著差异下调表达的基因涉及63个基因簇。第三部分,为了考察生物脱毒关键基因转化酚醛抑制物的能力,本研究通过遗传工程强化改造了底盘微生物Z. mobilis ZM4。通过强化Z. mobilis ZM4自身的酚醛抑制物还原代谢途径和重构酚醛抑制物氧化代谢途径,本研究尝试提高Z. mobilis ZM4转化酚醛抑制物和发酵纤维素乙醇的能力。结果表明,Pseudomonas putida KT2440来源的NAD+依赖型的醛脱氢酶(PP_2680)显著提高了Z. mobilis ZM4转化醛类抑制物和发酵纤维素乙醇的能力。PP_2680重组菌株在15%(w/w)固含量玉米秸秆水解液发酵24 h的乙醇浓度、乙醇产率和乙醇得率分别较对照菌株提高63.7%、100.0%和106.3%。PP 2680蛋白在离体条件下不具备还原能力,但是其依赖NAD(P)+氧化4-羟基苯甲醛、香草醛、糠醛和5-羟甲基糠醛生成4-羟基苯甲酸、香草酸、糠酸和2,5-呋喃二甲醛。但是,PP 2680在Z. mobilis ZM4活体条件下虽然提高了醛类抑制物转化和纤维素乙醇发酵的能力,但是未能氧化酚醛和呋喃醛产生相应的酸。而且,PP_2680 (NAD+-ALDH)和ZM01696(NADH-ADH)共表达间接证明了辅因子回补是PP 2680提高Z. mobilis ZM4醛类抑制物转化和纤维素乙醇发酵能力的重要原因。一个意外的发现是,PP 2680在Z. mobilisZM4的异源表达提高了ED途径的醇脱氢酶和氧化磷酸化过程H+转运ATPase、焦磷酸酶和细胞色素bd复合体编码基因的表达水平。第四部分,针对木质纤维素来源抑制物生物脱毒菌株理性改造的必要性,本研究提出构建木质纤维素来源抑制物的生物脱毒关键基因元器件库。主要依据抑制物胁迫条件下的转录组数据,本研究构建了细菌(B. subtilis、C. beijerinckii、C. glutamicum、E. coli、 Z. brevis、P. putida、T. pseudethanolicus和Z. mobilis)、酵母(Pichia stipites和S. cerevisiae)和霉菌(A. resinae)的酚类、呋喃类和弱酸类抑制物生物脱毒的关键基因元器件库。研究发现,酚类抑制物生物脱毒的关键基因主要涉及氧化还原、转运和调控；呋喃类抑制物生物脱毒的关键基因主要涉及氧化还原、转运、调控和氧化胁迫；乙酸生物脱毒的关键基因主要涉及中心碳代谢、转运和调控；成分复杂的木质纤维素水解液体系除了涉及转运和调控外,还涉及其他多方面的生物学过程。同时,基于构建的基因元器件库,本研究推测了Z. mobilis ZM4终极降解酚醛抑制物的代谢途径,揭示了酚醛抑制物氧化产酸(诸如原儿茶酸和3-O-甲基没食子酸)途径是Z. mobilis ZM4终极降解酚醛抑制物的关键代谢节点。综上所述,本研究从分子生物学水平解析了生物炼制脱毒菌株和发酵菌株转化酚醛抑制物的机制,测试了经代谢强化改造的Z. mobilis ZM4转化酚醛抑制物和发酵纤维素乙醇的能力,构建了细菌、酵母和霉菌的酚类、呋喃类和弱酸类生物脱毒的关键基因元器件库。本研究为生物炼制菌株的抑制物生物脱毒改造和发酵性能强化提供了重要的基因元器件库和整合生物加工平台。
[Abstract]:the phenolic inhibitor is one of the main inhibitors produced in the pre-treatment step of the lignocellulosic bio-refining process, which is mainly due to the excessive degradation of the lignin, which is representative of 4-hydroxybenzaldehyde belonging to the p-hydroxyphenyl group, Caryophyllaldehyde and guaiacol base class of caryophyllaldehyde. And the aromatic ring structure of the phenolic inhibitor is slow in degradation due to the difference of the inhibitor of the heavy acid (furfural and 5-hydroxymethylfurfural) and the weak acid (acetic acid, formic acid and ethyl-methyl-propionic acid). Therefore, the phenolic inhibitor is the main inhibitor of the cellulase and the fermentation microorganism. Biological detoxification is a brand-new detoxification concept and is thought to be an essential step in the process of future biorefinery. The main idea is to utilize the microorganisms with biodegradability to transform the inhibitor produced by the process of the lignocellulose pretreatment. At present, the study of the mechanism of biological detoxification of the organic acid and the organic acid is clear. The phenol-formaldehyde inhibitor is difficult to be quantitatively and quantitatively determined because the water-solubility difference is poor, and therefore, the biological detoxification mechanism of the phenolic inhibitor is less. In this study, RNA-Seq technique and DNA chip technology were used to analyze the metabolic pathway and the tolerance mechanism of the phenol-formaldehyde inhibitor of the biorefinery strain, Amphothecia resinae ZN1 and Zymomonas mobilis ZM4, and the key gene of the biological detoxification of the phenolic inhibitor was screened. and the ability of the phenolic inhibitor to be biologically detoxified and the whole biological processing strain of the cellulose ethanol fermentation is preliminarily tested and the phenols of the bacteria, the yeast and the mould are constructed according to the data of the transcription group, The key gene component library of the biological detoxication of the inhibitor of the weak acid type and the weak acid. In the first part, in order to explore the mechanism of the transformation of phenolic inhibitor from the mycobacterial A. resinae ZN1, A. resinae ZN1 virus-free phenolic inhibitor was examined by RNA-Seq technique to produce the transcription group of phenolic acid and phenolic alcohol. The results showed that 534,1576 and 1261 genes were significantly different in the transformation of 4-hydroxybenzaldehyde, syringaldehyde and vanillin, respectively. GO analysis found that redox and transport were the main biological processes of A. resinae ZN1 virus-free phenolic inhibitor. Based on the presumed metabolic pathway of A. resinae ZN1 to the conversion of the phenolic inhibitor, the study found that the alcohol dehydrogenase, the base alcohol dehydrogenase and the aldose reductase are the key enzymes for the reduction of the alcohol metabolism pathway of the phenolic inhibitor of A. resinae ZN1, and the aldehyde dehydrogenase is a key enzyme for the production of the acid metabolic pathway of the A. resinae ZN1 oxidation phenolic inhibitor. In the second part, in order to explore the mechanism of the production of ethanol-producing bacteria Z. mobiliis ZM4, this study investigated the transcriptome of the production of alcohol by Z. mobilis ZM4 by DNA chip technology. The results showed that 442,67 and 306 genes were differentially expressed under the stress of 4-hydroxybenzaldehyde, syringaldehyde and vanillin. The reduction, transport and control are the main molecular mechanism of the Z. mobilis ZM4 reduction phenolic inhibitor. This study identified 72 key genes of the Zmobiliis ZM4 reduction phenolic inhibitor, including the significant difference in the expression of ZMO1116 (Oxidioredoxymethyl) and ZMO1885 (NADH: flavin oxidoreductase/ NADH oxidase) under the stress of the phenolic inhibitor. At the same time, the present study found that the expression of 272 genes involved in the up-regulation of at least two phenolic inhibitors involved 36 gene clusters, and the down-regulation of 560 genes involved 63 gene clusters. In the third part, in order to investigate the ability of the biotoxin-free key gene to transform the phenolic inhibitor, this study has transformed the chassis microorganism Z. mobilis ZM4 by genetic engineering. This study attempts to improve the ability of Z. mobilis ZM4 to convert phenolic inhibitor and fermented cellulose ethanol by strengthening Z. mobilis ZM4 's own phenolic inhibitor to reduce its metabolic pathway and to reconstruct the pathway of oxidation and metabolism of phenolic inhibitor. The results showed that the NAD +-dependent aldehyde dehydrogenase (PP _ 2680) from Pseudomonas putida KT2440 significantly increased the ability of Z. mobiliis ZM4 to transform the aldehyde inhibitor and to ferment the cellulose ethanol. The ethanol concentration, ethanol yield and ethanol yield of the recombinant strain of PP _ 2680 were increased by 63.7%, 100.0% and 106.3%, respectively, under the condition of 15% (w/ w) and the yield of ethanol and the yield of ethanol were increased by 63.7%, 100.0% and 106.3%, respectively. Furfural and 5-hydroxymethylfurfural produce 4-hydroxybenzoic acid, vanillic acid, furfuryl acid and 2,5-hydroxydiformin. However, while the ability of the aldehyde-inhibitor conversion and the cellulose-ethanol fermentation is improved under the conditions of Z. mobiliis ZM4, the corresponding acid can not be produced by oxidizing the phenolic aldehyde and the calcitral. In addition, co-expression of the co-expression of PP _ 2680 (NAD +-ALDH) and ZM01696 (NADH-ADH) indirectly demonstrated that the co-expression of the cofactor is an important reason for the improvement of the transformation of the Z. mobiliis ZM4 aldehyde inhibitor and the ability of the cellulose ethanol to be fermented by the PP 2680. An unexpected finding is that the heterologous expression of PP 2680 in Z. mobiliisZM4 increases the level of expression of the alcohol dehydrogenase and the oxidative phosphorylation process H + transport ATPase, pyrophosphatase, and cytochrome bd complex encoding genes of the ED pathway. In the fourth part, aiming at the necessity of the rational transformation of the biological detoxification strain of the lignocellulose-derived inhibitor, the research puts forward the key gene component library for the biological detoxification of the wood-cellulose-derived inhibitor. In this study, the phenols of bacteria (B. subtilis, C. bejerinckii, C. gluttamicum, E. coli, Z. brevis, P. putida, T. pseudoanolicus and Z. mobilis), yeast (Pichia stipsites and S.cerevisiae) and mold (A. resineae) were constructed according to the data of the transcription group under the control of the inhibitor. The key gene component library of the biological detoxication of the inhibitor of the weak acid type and the weak acid. The research shows that the key genes of the biological detoxification of the phenolic inhibitor mainly relate to the oxidation reduction, the transport and the regulation, and the key genes of the biological detoxification of the phenolic inhibitor mainly relate to the oxidation reduction, the transport, the regulation and the oxidative stress, and the key genes of the biological detoxification of the acetic acid mainly relate to the central carbon metabolism, Transport and control; the complex lignocellulosic hydrolysate is involved in many other biological processes in addition to transport and control. At the same time, based on the constructed gene component library, this study has speculated that Z. mobilis ZM4 is the ultimate metabolic pathway for the ultimate degradation of the phenolic inhibitor. A key metabolic node for the ultimate degradation of the phenolic inhibitor of Z. mobiliis ZM4 is disclosed by the oxidative production of phenolic inhibitors (such as protocatechuic acid and 3-O-methylgallic acid). To sum up, this study analyzed the mechanism of the biorefinery detoxification strain and the fermentation strain to convert the phenolic inhibitor from the level of molecular biology, and tested the ability of the modified Z. mobilis ZM4 to convert the phenolic inhibitor and the fermented cellulose ethanol to construct the bacteria. The key gene component library of the biological detoxification of the phenols, the yeast and the weak acids of the yeast and the mould. The research provides an important gene component library and a whole biological processing platform for the biological detoxification transformation and the fermentation performance enhancement of the inhibitor of the biorefinery strain.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ223.122
本文编号：2488584
[Abstract]:the phenolic inhibitor is one of the main inhibitors produced in the pre-treatment step of the lignocellulosic bio-refining process, which is mainly due to the excessive degradation of the lignin, which is representative of 4-hydroxybenzaldehyde belonging to the p-hydroxyphenyl group, Caryophyllaldehyde and guaiacol base class of caryophyllaldehyde. And the aromatic ring structure of the phenolic inhibitor is slow in degradation due to the difference of the inhibitor of the heavy acid (furfural and 5-hydroxymethylfurfural) and the weak acid (acetic acid, formic acid and ethyl-methyl-propionic acid). Therefore, the phenolic inhibitor is the main inhibitor of the cellulase and the fermentation microorganism. Biological detoxification is a brand-new detoxification concept and is thought to be an essential step in the process of future biorefinery. The main idea is to utilize the microorganisms with biodegradability to transform the inhibitor produced by the process of the lignocellulose pretreatment. At present, the study of the mechanism of biological detoxification of the organic acid and the organic acid is clear. The phenol-formaldehyde inhibitor is difficult to be quantitatively and quantitatively determined because the water-solubility difference is poor, and therefore, the biological detoxification mechanism of the phenolic inhibitor is less. In this study, RNA-Seq technique and DNA chip technology were used to analyze the metabolic pathway and the tolerance mechanism of the phenol-formaldehyde inhibitor of the biorefinery strain, Amphothecia resinae ZN1 and Zymomonas mobilis ZM4, and the key gene of the biological detoxification of the phenolic inhibitor was screened. and the ability of the phenolic inhibitor to be biologically detoxified and the whole biological processing strain of the cellulose ethanol fermentation is preliminarily tested and the phenols of the bacteria, the yeast and the mould are constructed according to the data of the transcription group, The key gene component library of the biological detoxication of the inhibitor of the weak acid type and the weak acid. In the first part, in order to explore the mechanism of the transformation of phenolic inhibitor from the mycobacterial A. resinae ZN1, A. resinae ZN1 virus-free phenolic inhibitor was examined by RNA-Seq technique to produce the transcription group of phenolic acid and phenolic alcohol. The results showed that 534,1576 and 1261 genes were significantly different in the transformation of 4-hydroxybenzaldehyde, syringaldehyde and vanillin, respectively. GO analysis found that redox and transport were the main biological processes of A. resinae ZN1 virus-free phenolic inhibitor. Based on the presumed metabolic pathway of A. resinae ZN1 to the conversion of the phenolic inhibitor, the study found that the alcohol dehydrogenase, the base alcohol dehydrogenase and the aldose reductase are the key enzymes for the reduction of the alcohol metabolism pathway of the phenolic inhibitor of A. resinae ZN1, and the aldehyde dehydrogenase is a key enzyme for the production of the acid metabolic pathway of the A. resinae ZN1 oxidation phenolic inhibitor. In the second part, in order to explore the mechanism of the production of ethanol-producing bacteria Z. mobiliis ZM4, this study investigated the transcriptome of the production of alcohol by Z. mobilis ZM4 by DNA chip technology. The results showed that 442,67 and 306 genes were differentially expressed under the stress of 4-hydroxybenzaldehyde, syringaldehyde and vanillin. The reduction, transport and control are the main molecular mechanism of the Z. mobilis ZM4 reduction phenolic inhibitor. This study identified 72 key genes of the Zmobiliis ZM4 reduction phenolic inhibitor, including the significant difference in the expression of ZMO1116 (Oxidioredoxymethyl) and ZMO1885 (NADH: flavin oxidoreductase/ NADH oxidase) under the stress of the phenolic inhibitor. At the same time, the present study found that the expression of 272 genes involved in the up-regulation of at least two phenolic inhibitors involved 36 gene clusters, and the down-regulation of 560 genes involved 63 gene clusters. In the third part, in order to investigate the ability of the biotoxin-free key gene to transform the phenolic inhibitor, this study has transformed the chassis microorganism Z. mobilis ZM4 by genetic engineering. This study attempts to improve the ability of Z. mobilis ZM4 to convert phenolic inhibitor and fermented cellulose ethanol by strengthening Z. mobilis ZM4 's own phenolic inhibitor to reduce its metabolic pathway and to reconstruct the pathway of oxidation and metabolism of phenolic inhibitor. The results showed that the NAD +-dependent aldehyde dehydrogenase (PP _ 2680) from Pseudomonas putida KT2440 significantly increased the ability of Z. mobiliis ZM4 to transform the aldehyde inhibitor and to ferment the cellulose ethanol. The ethanol concentration, ethanol yield and ethanol yield of the recombinant strain of PP _ 2680 were increased by 63.7%, 100.0% and 106.3%, respectively, under the condition of 15% (w/ w) and the yield of ethanol and the yield of ethanol were increased by 63.7%, 100.0% and 106.3%, respectively. Furfural and 5-hydroxymethylfurfural produce 4-hydroxybenzoic acid, vanillic acid, furfuryl acid and 2,5-hydroxydiformin. However, while the ability of the aldehyde-inhibitor conversion and the cellulose-ethanol fermentation is improved under the conditions of Z. mobiliis ZM4, the corresponding acid can not be produced by oxidizing the phenolic aldehyde and the calcitral. In addition, co-expression of the co-expression of PP _ 2680 (NAD +-ALDH) and ZM01696 (NADH-ADH) indirectly demonstrated that the co-expression of the cofactor is an important reason for the improvement of the transformation of the Z. mobiliis ZM4 aldehyde inhibitor and the ability of the cellulose ethanol to be fermented by the PP 2680. An unexpected finding is that the heterologous expression of PP 2680 in Z. mobiliisZM4 increases the level of expression of the alcohol dehydrogenase and the oxidative phosphorylation process H + transport ATPase, pyrophosphatase, and cytochrome bd complex encoding genes of the ED pathway. In the fourth part, aiming at the necessity of the rational transformation of the biological detoxification strain of the lignocellulose-derived inhibitor, the research puts forward the key gene component library for the biological detoxification of the wood-cellulose-derived inhibitor. In this study, the phenols of bacteria (B. subtilis, C. bejerinckii, C. gluttamicum, E. coli, Z. brevis, P. putida, T. pseudoanolicus and Z. mobilis), yeast (Pichia stipsites and S.cerevisiae) and mold (A. resineae) were constructed according to the data of the transcription group under the control of the inhibitor. The key gene component library of the biological detoxication of the inhibitor of the weak acid type and the weak acid. The research shows that the key genes of the biological detoxification of the phenolic inhibitor mainly relate to the oxidation reduction, the transport and the regulation, and the key genes of the biological detoxification of the phenolic inhibitor mainly relate to the oxidation reduction, the transport, the regulation and the oxidative stress, and the key genes of the biological detoxification of the acetic acid mainly relate to the central carbon metabolism, Transport and control; the complex lignocellulosic hydrolysate is involved in many other biological processes in addition to transport and control. At the same time, based on the constructed gene component library, this study has speculated that Z. mobilis ZM4 is the ultimate metabolic pathway for the ultimate degradation of the phenolic inhibitor. A key metabolic node for the ultimate degradation of the phenolic inhibitor of Z. mobiliis ZM4 is disclosed by the oxidative production of phenolic inhibitors (such as protocatechuic acid and 3-O-methylgallic acid). To sum up, this study analyzed the mechanism of the biorefinery detoxification strain and the fermentation strain to convert the phenolic inhibitor from the level of molecular biology, and tested the ability of the modified Z. mobilis ZM4 to convert the phenolic inhibitor and the fermented cellulose ethanol to construct the bacteria. The key gene component library of the biological detoxification of the phenols, the yeast and the weak acids of the yeast and the mould. The research provides an important gene component library and a whole biological processing platform for the biological detoxification transformation and the fermentation performance enhancement of the inhibitor of the biorefinery strain.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ223.122
【参考文献】
相关硕士学位论文 前1条
1 涂毅;木质纤维素依赖型Pediococcus acidilactici DQ2中外源功能基因的表达及基因敲除[D];华东理工大学;2013年
，本文编号：2488584
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