【摘要】：酚醛抑制物是木質(zhì)纖維素生物煉制過程的預(yù)處理步驟產(chǎn)生的主要抑制物之一,其主要源于木質(zhì)素的過度降解,其代表性化合物包括隸屬p-羥基苯基類的4-羥基苯甲醛、丁香基類的丁香醛和愈創(chuàng)木酚基類的香草醛。與呋喃類(糠醛和5-羥甲基糠醛)和弱酸類(乙酸、甲酸和乙酰丙酸)抑制物不同,種類較多的酚醛抑制物難揮發(fā)且水溶性較差,其芳香環(huán)結(jié)構(gòu)致使其降解緩慢。因此,酚醛抑制物是纖維素酶和發(fā)酵微生物的主要抑制物。生物脫毒是一種全新的脫毒概念,被認(rèn)為是未來生物煉制過程不可或缺的步驟,其主要思路是利用具有生物降解能力的微生物轉(zhuǎn)化木質(zhì)纖維素預(yù)處理過程產(chǎn)生的抑制物。目前,對呋喃醛和有機(jī)酸生物脫毒機(jī)理的研究比較清楚。酚醛抑制物因?yàn)樗苄圆疃y以精確定性定量,因此其生物脫毒機(jī)理報道較少。本研究采用RNA-Seq技術(shù)和DNA芯片技術(shù),對生物煉制菌株樹脂枝孢霉菌(Amorphotheca resinae ZN1)和運(yùn)動發(fā)酵單胞菌(Zymomonas mobilis ZM4)的酚醛抑制物代謝途徑和耐受機(jī)制進(jìn)行了解析；對酚醛抑制物生物脫毒關(guān)鍵基因進(jìn)行了篩選,并在運(yùn)動發(fā)酵單胞菌進(jìn)行了酚醛抑制物代謝途徑的強(qiáng)化改造,初步測試了酚醛抑制物生物脫毒與纖維素乙醇發(fā)酵的整合生物加工菌株的能力；主要依據(jù)轉(zhuǎn)錄組數(shù)據(jù),構(gòu)建了細(xì)菌、酵母和霉菌的酚類、呋喃類和弱酸類抑制物生物脫毒的關(guān)鍵基因元器件庫。第一部分,為了探究專司生物脫毒的絲狀真菌A.resinae ZN1轉(zhuǎn)化酚醛抑制物的機(jī)制,本研究通過RNA-Seq技術(shù)考察了A. resinae ZN1脫毒酚醛抑制物生成酚酸和酚醇的轉(zhuǎn)錄組。結(jié)果發(fā)現(xiàn),534個、1576個和1261個基因分別在4-羥基苯甲醛、丁香醛和香草醛轉(zhuǎn)化過程中顯著差異表達(dá)。GO分析發(fā)現(xiàn),氧化還原和轉(zhuǎn)運(yùn)是A. resinae ZN1脫毒酚醛抑制物的主要生物學(xué)過程�；谕茰y的A. resinae ZN1轉(zhuǎn)化酚醛抑制物的代謝途徑,本研究發(fā)現(xiàn)醇脫氫酶、酰基醇脫氫酶和醛還原酶是A. resinae ZN1還原酚醛抑制物產(chǎn)醇代謝途徑的關(guān)鍵酶,醛脫氫酶是A. resinae ZN1氧化酚醛抑制物產(chǎn)酸代謝途徑的關(guān)鍵酶。第二部分,為了探究產(chǎn)乙醇細(xì)菌Z. mobilis ZM4轉(zhuǎn)化酚醛抑制物的機(jī)制,本研究通過DNA芯片技術(shù)考察了Z. mobilis ZM4還原酚醛抑制物產(chǎn)醇的轉(zhuǎn)錄組。結(jié)果發(fā)現(xiàn),442個、67個和306個基因分別在4-羥基苯甲醛、丁香醛和香草醛脅迫下顯著差異表達(dá)。還原、轉(zhuǎn)運(yùn)和調(diào)控是Z. mobilis ZM4還原酚醛抑制物的主要分子機(jī)制。本研究鑒定了Zmobilis ZM4還原酚醛抑制物的72個關(guān)鍵基因,其中包括酚醛抑制物脅迫下均顯著差異上調(diào)表達(dá)的ZMO1116 (Oxidoreductase)和ZMO1885 (NADH:flavin oxidoreductase/NADH oxidase)。同時,本研究通過基因組圖譜定位發(fā)現(xiàn),在至少2種酚醛抑制物脅迫下顯著差異上調(diào)表達(dá)的272個基因涉及36個基因簇,560個顯著差異下調(diào)表達(dá)的基因涉及63個基因簇。第三部分,為了考察生物脫毒關(guān)鍵基因轉(zhuǎn)化酚醛抑制物的能力,本研究通過遺傳工程強(qiáng)化改造了底盤微生物Z. mobilis ZM4。通過強(qiáng)化Z. mobilis ZM4自身的酚醛抑制物還原代謝途徑和重構(gòu)酚醛抑制物氧化代謝途徑,本研究嘗試提高Z. mobilis ZM4轉(zhuǎn)化酚醛抑制物和發(fā)酵纖維素乙醇的能力。結(jié)果表明,Pseudomonas putida KT2440來源的NAD+依賴型的醛脫氫酶(PP_2680)顯著提高了Z. mobilis ZM4轉(zhuǎn)化醛類抑制物和發(fā)酵纖維素乙醇的能力。PP_2680重組菌株在15%(w/w)固含量玉米秸稈水解液發(fā)酵24 h的乙醇濃度、乙醇產(chǎn)率和乙醇得率分別較對照菌株提高63.7%、100.0%和106.3%。PP 2680蛋白在離體條件下不具備還原能力,但是其依賴NAD(P)+氧化4-羥基苯甲醛、香草醛、糠醛和5-羥甲基糠醛生成4-羥基苯甲酸、香草酸、糠酸和2,5-呋喃二甲醛。但是,PP 2680在Z. mobilis ZM4活體條件下雖然提高了醛類抑制物轉(zhuǎn)化和纖維素乙醇發(fā)酵的能力,但是未能氧化酚醛和呋喃醛產(chǎn)生相應(yīng)的酸。而且,PP_2680 (NAD+-ALDH)和ZM01696(NADH-ADH)共表達(dá)間接證明了輔因子回補(bǔ)是PP 2680提高Z. mobilis ZM4醛類抑制物轉(zhuǎn)化和纖維素乙醇發(fā)酵能力的重要原因。一個意外的發(fā)現(xiàn)是,PP 2680在Z. mobilisZM4的異源表達(dá)提高了ED途徑的醇脫氫酶和氧化磷酸化過程H+轉(zhuǎn)運(yùn)ATPase、焦磷酸酶和細(xì)胞色素bd復(fù)合體編碼基因的表達(dá)水平。第四部分,針對木質(zhì)纖維素來源抑制物生物脫毒菌株理性改造的必要性,本研究提出構(gòu)建木質(zhì)纖維素來源抑制物的生物脫毒關(guān)鍵基因元器件庫。主要依據(jù)抑制物脅迫條件下的轉(zhuǎn)錄組數(shù)據(jù),本研究構(gòu)建了細(xì)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)的酚類、呋喃類和弱酸類抑制物生物脫毒的關(guān)鍵基因元器件庫。研究發(fā)現(xiàn),酚類抑制物生物脫毒的關(guān)鍵基因主要涉及氧化還原、轉(zhuǎn)運(yùn)和調(diào)控；呋喃類抑制物生物脫毒的關(guān)鍵基因主要涉及氧化還原、轉(zhuǎn)運(yùn)、調(diào)控和氧化脅迫；乙酸生物脫毒的關(guān)鍵基因主要涉及中心碳代謝、轉(zhuǎn)運(yùn)和調(diào)控；成分復(fù)雜的木質(zhì)纖維素水解液體系除了涉及轉(zhuǎn)運(yùn)和調(diào)控外,還涉及其他多方面的生物學(xué)過程。同時,基于構(gòu)建的基因元器件庫,本研究推測了Z. mobilis ZM4終極降解酚醛抑制物的代謝途徑,揭示了酚醛抑制物氧化產(chǎn)酸(諸如原兒茶酸和3-O-甲基沒食子酸)途徑是Z. mobilis ZM4終極降解酚醛抑制物的關(guān)鍵代謝節(jié)點(diǎn)。綜上所述,本研究從分子生物學(xué)水平解析了生物煉制脫毒菌株和發(fā)酵菌株轉(zhuǎn)化酚醛抑制物的機(jī)制,測試了經(jīng)代謝強(qiáng)化改造的Z. mobilis ZM4轉(zhuǎn)化酚醛抑制物和發(fā)酵纖維素乙醇的能力,構(gòu)建了細(xì)菌、酵母和霉菌的酚類、呋喃類和弱酸類生物脫毒的關(guān)鍵基因元器件庫。本研究為生物煉制菌株的抑制物生物脫毒改造和發(fā)酵性能強(qiáng)化提供了重要的基因元器件庫和整合生物加工平臺。
[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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：TQ223.122

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相關(guān)碩士學(xué)位論文 前1條

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本文編號：2488584