呋喃醛抑制物的生物脱毒机理解析及高抗逆生物炼制发酵工程菌株的构建

发布时间：2018-06-19 21:23

  本文选题：木质纤维素 + 呋喃醛　； 参考：《华东理工大学》2017年博士论文

【摘要】：糠醛、5-羟甲基糠醛是两种典型的呋喃醛抑制物,二者在木质纤维素预处理过程中分别由戊糖和己糖的降解产生,可对后续生物炼制发酵菌株的生长和发酵产生强烈的抑制作用。在预处理物料中糠醛、5-羟甲基糠醛的含量较高、抑制作用较强,因此,消除糠醛、5-羟甲基糠醛对发酵微生物的抑制作用被认为是木质纤维素生物炼制工艺的关键环节。借助某些特定的微生物在发酵前对预处理后物料中所含有的呋喃醛抑制物进行生物转化("生物脱毒")是缓解或消除抑制物抑制作用的有效方式,而使发酵微生物具备较高的呋喃醛抑制物转化性能或耐受性能也是提高生物炼制效率的重要研究课题。树脂枝孢霉Amorphotheca resinae ZN1是在实验室前期工作中筛选得到的具有强大生物脱毒功能的生物脱毒菌株,该菌株已经在木质纤维素生物炼制生产乙醇、油脂、乳酸、柠檬酸、葡萄糖酸、木糖酸等产品的过程中得到了广泛应用并开始了产业化示范应用。本论文对A.resinae ZN1中呋喃醛抑制物的降解机理进行了研究。首先,采用定量PCR技术对A.resinae ZN1中与抑制物转化相关的137个基因在呋喃醛和呋喃醇胁迫下的转录水平进行了定量,证实A.resinaeZN1的多个醇脱氢酶、醛还原酶或醛酮还原酶可还原呋喃醛(糠醛、5-羟甲基糠醛)生成相应的呋喃醇(糠醇、羟甲基糠醇),而生成的呋喃醇在相应的醇脱氢酶、醛脱氢酶和氧化酶催化下进一步氧化生成相应的呋喃酸(糠酸、羟甲基糠酸)。其次,采用RNA-Seq技术在呋喃醛胁迫下对A.resinae ZN1进行了全基因转录水平分析,发现A.resinae ZN1除了可诱导胞内相关的氧化还原酶基因上调表达,还会诱导胞内与氧化还原力生成(三羧酸循环)和能量生成(呼吸链)相关的基因上调表达,以促进呋喃醛的降解转化。转录组中差异表达基因的分析表明,呋喃醛的降解还涉及物质转运、氧化胁迫应激反应等过程,这些生物学过程在A.resinae ZN1降解呋喃醛抑制物的过程中发挥了重要作用。运动发酵单胞菌Zymomonas mobilis ZM4是纤维素乙醇发酵生产的主要工业菌株之一。Z.mobilis ZM4自身可将低浓度的糠醛、5-羟甲基糠醛转化为相应的糠醇、羟甲基糠醇,但细胞自身的生长和代谢会受到呋喃醛抑制物的抑制。在Z.mobilis ZM4中过表达与呋喃醛转化相关的基因,包括来自A.resinae ZN1的外源醛酮还原酶基因ARZ__13395_T1或来自Z.mobilis ZM4自身的醇脱氢酶基因ZM01771时,Z.mobilis ZM4转化呋喃醛的能力得到明显增强,并促进了菌体在玉米秸秆水解液中的细胞生长、葡萄糖消耗和乙醇生成。进一步在过表达ZM01771的菌株中共表达与还原力(NADPH或NADH)供给相关的基因。ZM01771编码蛋白以NADPH为辅因子,当共表达来自E.coli的转氢酶基因udhA时,可将胞内的NADH转化为NADPH用于ZM01771对呋喃醛的转化,进一步提高了菌体对呋喃醛抑制物的转化能力。谷氨酸棒杆菌Corynebacterium glutamicum S9114-128是在实验室前期工作中经玉米秸秆水解液长期驯化后得到的一株谷氨酸发酵菌株,具有明显高于野生菌株的呋喃醛抑制物耐受性能。为解析C.glutamicumS9114-128抑制物耐受性能增强的分子机理,对其进行基因组重测序并对测序得到的相关突变基因进行功能验证分析。结果显示,位于两个基因(CGS9114_RS11050和CGS9114__RS11055)间区的SNP突变使得与葡萄糖转运相关的基因CGS9114_RS11050(ptsI)上调表达,促进了葡萄糖向胞内的转运;葡萄糖转运的加快在促进葡萄糖代谢的同时也使得部分葡萄糖偏向乳酸生成;位于α-酮戊二酸脱羧酶基因CGS9114_RS03450(odhA)处的单碱基缺失突变使得驯化菌株中的TCA循环代谢增强,谷氨酸积累下降。然而对于C.glutamicum S9114-128中观察到的代谢流偏移现象与抑制物耐受性能增强之间的联系还仍有待于进一步分析确认。本论文针对呋喃醛抑制物对生物炼制微生物的抑制,提出了生物脱毒的应对策略和构建高抑制物耐受型发酵菌株的应对策略。通过上述研究,阐明了生物脱毒真菌A.resinae ZN1降解呋喃醛抑制物的分子生物学机理,并得到了与抑制物转化相关的关键作用基因;对乙醇发酵菌株Z.mobilis ZM4的呋喃醛抑制物转化进行了代谢工程改造,有效地提高了菌株对抑制物的耐受性能;对谷氨酸发酵菌株C.glutamicum S9114-128驯化菌株的基因组突变位点进行分析,初步解析了该发酵菌株抑制物耐受性能提高的分子机理。上述研究结果将为木质纤维素生物炼制的工业化推广和应用奠定理论基础。
[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.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.6
，

本文编号：2041332