發(fā)布時(shí)間：2018-06-19 21:23

  本文選題：木質(zhì)纖維素 + 呋喃醛��； 參考：《華東理工大學(xué)》2017年博士論文

【摘要】：糠醛、5-羥甲基糠醛是兩種典型的呋喃醛抑制物,二者在木質(zhì)纖維素預(yù)處理過(guò)程中分別由戊糖和己糖的降解產(chǎn)生,可對(duì)后續(xù)生物煉制發(fā)酵菌株的生長(zhǎng)和發(fā)酵產(chǎn)生強(qiáng)烈的抑制作用。在預(yù)處理物料中糠醛、5-羥甲基糠醛的含量較高、抑制作用較強(qiáng),因此,消除糠醛、5-羥甲基糠醛對(duì)發(fā)酵微生物的抑制作用被認(rèn)為是木質(zhì)纖維素生物煉制工藝的關(guān)鍵環(huán)節(jié)。借助某些特定的微生物在發(fā)酵前對(duì)預(yù)處理后物料中所含有的呋喃醛抑制物進(jìn)行生物轉(zhuǎn)化("生物脫毒")是緩解或消除抑制物抑制作用的有效方式,而使發(fā)酵微生物具備較高的呋喃醛抑制物轉(zhuǎn)化性能或耐受性能也是提高生物煉制效率的重要研究課題。樹(shù)脂枝孢霉Amorphotheca resinae ZN1是在實(shí)驗(yàn)室前期工作中篩選得到的具有強(qiáng)大生物脫毒功能的生物脫毒菌株,該菌株已經(jīng)在木質(zhì)纖維素生物煉制生產(chǎn)乙醇、油脂、乳酸、檸檬酸、葡萄糖酸、木糖酸等產(chǎn)品的過(guò)程中得到了廣泛應(yīng)用并開(kāi)始了產(chǎn)業(yè)化示范應(yīng)用。本論文對(duì)A.resinae ZN1中呋喃醛抑制物的降解機(jī)理進(jìn)行了研究。首先,采用定量PCR技術(shù)對(duì)A.resinae ZN1中與抑制物轉(zhuǎn)化相關(guān)的137個(gè)基因在呋喃醛和呋喃醇脅迫下的轉(zhuǎn)錄水平進(jìn)行了定量,證實(shí)A.resinaeZN1的多個(gè)醇脫氫酶、醛還原酶或醛酮還原酶可還原呋喃醛(糠醛、5-羥甲基糠醛)生成相應(yīng)的呋喃醇(糠醇、羥甲基糠醇),而生成的呋喃醇在相應(yīng)的醇脫氫酶、醛脫氫酶和氧化酶催化下進(jìn)一步氧化生成相應(yīng)的呋喃酸(糠酸、羥甲基糠酸)。其次,采用RNA-Seq技術(shù)在呋喃醛脅迫下對(duì)A.resinae ZN1進(jìn)行了全基因轉(zhuǎn)錄水平分析,發(fā)現(xiàn)A.resinae ZN1除了可誘導(dǎo)胞內(nèi)相關(guān)的氧化還原酶基因上調(diào)表達(dá),還會(huì)誘導(dǎo)胞內(nèi)與氧化還原力生成(三羧酸循環(huán))和能量生成(呼吸鏈)相關(guān)的基因上調(diào)表達(dá),以促進(jìn)呋喃醛的降解轉(zhuǎn)化。轉(zhuǎn)錄組中差異表達(dá)基因的分析表明,呋喃醛的降解還涉及物質(zhì)轉(zhuǎn)運(yùn)、氧化脅迫應(yīng)激反應(yīng)等過(guò)程,這些生物學(xué)過(guò)程在A.resinae ZN1降解呋喃醛抑制物的過(guò)程中發(fā)揮了重要作用。運(yùn)動(dòng)發(fā)酵單胞菌Zymomonas mobilis ZM4是纖維素乙醇發(fā)酵生產(chǎn)的主要工業(yè)菌株之一。Z.mobilis ZM4自身可將低濃度的糠醛、5-羥甲基糠醛轉(zhuǎn)化為相應(yīng)的糠醇、羥甲基糠醇,但細(xì)胞自身的生長(zhǎng)和代謝會(huì)受到呋喃醛抑制物的抑制。在Z.mobilis ZM4中過(guò)表達(dá)與呋喃醛轉(zhuǎn)化相關(guān)的基因,包括來(lái)自A.resinae ZN1的外源醛酮還原酶基因ARZ__13395_T1或來(lái)自Z.mobilis ZM4自身的醇脫氫酶基因ZM01771時(shí),Z.mobilis ZM4轉(zhuǎn)化呋喃醛的能力得到明顯增強(qiáng),并促進(jìn)了菌體在玉米秸稈水解液中的細(xì)胞生長(zhǎng)、葡萄糖消耗和乙醇生成。進(jìn)一步在過(guò)表達(dá)ZM01771的菌株中共表達(dá)與還原力(NADPH或NADH)供給相關(guān)的基因。ZM01771編碼蛋白以NADPH為輔因子,當(dāng)共表達(dá)來(lái)自E.coli的轉(zhuǎn)氫酶基因udhA時(shí),可將胞內(nèi)的NADH轉(zhuǎn)化為NADPH用于ZM01771對(duì)呋喃醛的轉(zhuǎn)化,進(jìn)一步提高了菌體對(duì)呋喃醛抑制物的轉(zhuǎn)化能力。谷氨酸棒桿菌Corynebacterium glutamicum S9114-128是在實(shí)驗(yàn)室前期工作中經(jīng)玉米秸稈水解液長(zhǎng)期馴化后得到的一株谷氨酸發(fā)酵菌株,具有明顯高于野生菌株的呋喃醛抑制物耐受性能。為解析C.glutamicumS9114-128抑制物耐受性能增強(qiáng)的分子機(jī)理,對(duì)其進(jìn)行基因組重測(cè)序并對(duì)測(cè)序得到的相關(guān)突變基因進(jìn)行功能驗(yàn)證分析。結(jié)果顯示,位于兩個(gè)基因(CGS9114_RS11050和CGS9114__RS11055)間區(qū)的SNP突變使得與葡萄糖轉(zhuǎn)運(yùn)相關(guān)的基因CGS9114_RS11050(ptsI)上調(diào)表達(dá),促進(jìn)了葡萄糖向胞內(nèi)的轉(zhuǎn)運(yùn);葡萄糖轉(zhuǎn)運(yùn)的加快在促進(jìn)葡萄糖代謝的同時(shí)也使得部分葡萄糖偏向乳酸生成;位于α-酮戊二酸脫羧酶基因CGS9114_RS03450(odhA)處的單堿基缺失突變使得馴化菌株中的TCA循環(huán)代謝增強(qiáng),谷氨酸積累下降。然而對(duì)于C.glutamicum S9114-128中觀察到的代謝流偏移現(xiàn)象與抑制物耐受性能增強(qiáng)之間的聯(lián)系還仍有待于進(jìn)一步分析確認(rèn)。本論文針對(duì)呋喃醛抑制物對(duì)生物煉制微生物的抑制,提出了生物脫毒的應(yīng)對(duì)策略和構(gòu)建高抑制物耐受型發(fā)酵菌株的應(yīng)對(duì)策略。通過(guò)上述研究,闡明了生物脫毒真菌A.resinae ZN1降解呋喃醛抑制物的分子生物學(xué)機(jī)理,并得到了與抑制物轉(zhuǎn)化相關(guān)的關(guān)鍵作用基因;對(duì)乙醇發(fā)酵菌株Z.mobilis ZM4的呋喃醛抑制物轉(zhuǎn)化進(jìn)行了代謝工程改造,有效地提高了菌株對(duì)抑制物的耐受性能;對(duì)谷氨酸發(fā)酵菌株C.glutamicum S9114-128馴化菌株的基因組突變位點(diǎn)進(jìn)行分析,初步解析了該發(fā)酵菌株抑制物耐受性能提高的分子機(jī)理。上述研究結(jié)果將為木質(zhì)纖維素生物煉制的工業(yè)化推廣和應(yīng)用奠定理論基礎(chǔ)。
[Abstract]:Furfural, 5- hydroxymethyl furfural is two typical furan aldehyde inhibitor. The two are degraded by pentose and hexose respectively during the process of lignocellulose pretreatment, which can strongly inhibit the growth and fermentation of the fermentation strains. The content of furfural, 5- hydroxymethyl furfural in the pretreated materials is higher, and the inhibition effect is higher. Therefore, the elimination of furfural and the inhibition of 5- hydroxymethyl furfural on the fermentation microorganism is considered to be the key link in the biologic process of lignocellulose. With certain specific microorganisms, the biological conversion ("biological detoxification") of the furan aldehyde inhibitor contained in the pretreated materials ("biological detoxification") is a remission or elimination of inhibition by certain microorganisms before the fermentation. It is an important research topic to make the fermentation microorganism with high furan aldehyde inhibitor conversion performance or tolerance performance. The resin Cladosporium Amorphotheca resinae ZN1 is a biodetoxified strain with strong biological detoxification function screened in the early laboratory work. The strain has been widely used in the process of producing ethanol, oil, lactic acid, citric acid, gluconic acid, xylose acid and other products in lignocellulosic bioreaction, and began to demonstrate the application of industrialization. In this paper, the degradation mechanism of furaldehyde inhibitor in A.resinae ZN1 was studied. First, the quantitative PCR Technology was used for A.resinae ZN1. The transcriptional level of the 137 genes related to the transformation of inhibitors in furan aldehyde and furanol stress was quantified. It was confirmed that the multiple alcohol dehydrogenases of A.resinaeZN1, aldehyde reductase or aldehyde reductase can reduce furan aldehyde (furfural, 5- hydroxymethyl furfural) to produce furan alcohol (furfuryl alcohol, hydroxymethyl furfuryl alcohol), and the furan alcohol produced is corresponding. Under the catalysis of alcohol dehydrogenase, aldehyde dehydrogenase and oxidase, the corresponding furan acid (furfuric acid, hydroxymethyl furfuric acid) was further oxidized. Secondly, the whole gene transcription level of A.resinae ZN1 was analyzed under furan Aldehyde Stress by RNA-Seq technology. It was found that A.resinae ZN1 could induce the up-regulated expression of the intracellular related oxidoreductase gene. The analysis of the differential expression genes in the transcriptional group shows that the degradation of furaldehyde is also involved in the process of material transport and oxidative stress reaction, these biological processes are in A.resinae Z. N1 has played an important role in the degradation of furan aldehyde inhibitors. Zymomonas mobilis ZM4, one of the main industrial strains of cellulosic ethanol fermentation, is one of the major industrial strains of cellulosic ethanol,.Z.mobilis ZM4 itself can transform the low concentration furfural, 5- hydroxymethyl furfural into the corresponding furfuryl alcohol, hydroxymethyl furfuryl alcohol, but the growth and metabolism of the cell itself Inhibition of furaldehyde inhibitors. In Z.mobilis ZM4, the ability to transform furaldehyde with furaldehyde transformation related genes, including the exogenous aldosterone reductase gene ARZ__13395_T1 from A.resinae ZN1 or the alcohol dehydrogenase gene ZM01771 from Z.mobilis ZM4 itself, has been significantly enhanced and promoted the bacteria. Cell growth, glucose consumption and ethanol production in the hydrolysate of corn straw. Further, the gene.ZM01771 encoded protein associated with the supply of reductive force (NADPH or NADH) supplied by the strain expressing ZM01771 is supplemented by NADPH as a cofactor, and the intracellular NADH can be converted to NADPH when the co expression of the E.coli gene udhA is expressed. The transformation of furan aldehyde by ZM01771 further improves the transformation ability of the fungus to furan aldehyde inhibitor. Corynebacterium glutamicum S9114-128 of Bacillus glutamatosus is a strain of glutamic acid fermentation obtained by long-term domestication of corn straw hydrolysate in the early laboratory work, which is obviously higher than the furan aldehyde of the wild strain. In order to analyze the molecular mechanism of C.glutamicumS9114-128 inhibitor tolerance, genomic resequencing and functional verification of related mutations were carried out. The results showed that the SNP mutation located in the area between the two genes (CGS9114_RS11050 and CGS9114__RS11055) was associated with the glucose transport phase. The gene CGS9114_RS11050 (ptsI) up-regulated and promoted the transshipment of glucose to the intracellular; glucose transport accelerated while promoting glucose metabolism and partial glucose bias to lactic acid production; the single base deletion mutation located at the CGS9114_RS03450 (odhA) of the alpha ketopaldiacid decarboxylase gene resulted in the TCA in the domesticated strain. The relationship between the accumulation of glutamic acid and the increase of the metabolism of C.glutamicum S9114-128 remains to be further confirmed. In this study, the molecular biological mechanism of the biodetoxified fungus A.resinae ZN1 degradation of furaldehyde inhibitor was clarified, and the key gene related to the inhibitor transformation was obtained, and the conversion of the furaldehyde inhibitor for the ethanol fermented strain Z.mobilis ZM4 was carried out. The tolerance performance of the strain to inhibitor was effectively improved, and the mutation site of the C.glutamicum S9114-128 domesticated strain of glutamic acid fermentation strain was analyzed, and the molecular mechanism of improving the tolerance performance of the fermented strain was preliminarily analyzed. The results of the above study will be popularized for the industrialization of lignocellulose biorefinery. And the application lays the theoretical foundation.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ920.6
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本文編號(hào)：2041332