里氏木霉纖維素酶高產(chǎn)菌株遺傳改造及新型糖苷水解酶的挖掘
發(fā)布時間：2018-08-27 17:37
【摘要】：里氏木霉是生物質(zhì)降解酶的重要生產(chǎn)者之一,其生產(chǎn)的酶被廣泛應(yīng)用于工業(yè)生產(chǎn)。一些里氏木霉高產(chǎn)突變菌株已經(jīng)通過傳統(tǒng)誘變方法獲得。然而,生物質(zhì)降解酶生產(chǎn)的高成本仍然是其商業(yè)化應(yīng)用的巨大挑戰(zhàn)。此外,這些突變菌株相應(yīng)表型下的遺傳機制僅僅被部分地理解。全面地理解遺傳改變對纖維素酶生產(chǎn)的影響有利于開發(fā)更高效的纖維素酶生產(chǎn)菌株。在里氏木霉中,pH也是影響纖維素酶生產(chǎn)的重要因素。質(zhì)膜H~+-ATP酶在調(diào)節(jié)胞內(nèi)pH穩(wěn)態(tài)和營養(yǎng)攝取等生理過程中發(fā)揮著關(guān)鍵作用。然而,里氏木霉的質(zhì)膜H~+-ATP酶的功能到目前為止仍然沒有被研究。多種糖苷水解酶的組合使用已經(jīng)廣泛應(yīng)用于工業(yè)生產(chǎn)。為了滿足糖苷水解酶在工業(yè)生產(chǎn)中的需求,工業(yè)上迫切需要具有高表現(xiàn)力的糖苷水解酶來降低生產(chǎn)成本。宏基因組學(xué)已經(jīng)成為一種強有力的方法直接研究微生物群落的多樣性和挖掘新型生物催化劑。為了開發(fā)更高效的酶制劑生產(chǎn)菌株和挖掘新的糖苷水解酶,本文從以下三個方面展開了研究。1)一株里氏木霉突變菌株SS-Ⅱ通過多次的NTG誘變在菌株NG14的基礎(chǔ)上分離獲得。與菌株RUT-C30相比,擁有完整Cre1蛋白的SS-Ⅱ在微晶纖維素或乳糖培養(yǎng)下展現(xiàn)出更快的生長和約1.5倍高的羧甲基纖維素酶活性。通過SS-Ⅱ和RUT-C30的轉(zhuǎn)錄組數(shù)據(jù)的對比分析,我們發(fā)現(xiàn)有1764個基因在微晶纖維素、葡萄糖、乳糖和小麥秸稈下差異化表達(dá)。在此基礎(chǔ)上,65個纖維素降解相關(guān)的酶、41個轉(zhuǎn)錄因子和152個轉(zhuǎn)運蛋白的轉(zhuǎn)錄數(shù)據(jù)被進(jìn)一步分析。為了鑒定在SS-Ⅱ中發(fā)生的遺傳突變,我們對其基因組進(jìn)行了測序。在SS-Ⅱ中,總共有184個單核苷酸位點突變和40個小的插入/缺失被鑒定。此外,157個受突變影響的基因被鑒定。在這些基因中,大多數(shù)涉及運輸、分泌、蛋白質(zhì)代謝和轉(zhuǎn)錄。9個在SS-Ⅱ中受突變影響的基因被進(jìn)一步分析。微晶纖維素培養(yǎng)下,在RUT-C30中敲除其中的3個基因會明顯影響纖維素酶的生產(chǎn)。菌株SS-Ⅱ和RUT-C30的轉(zhuǎn)錄組比較分析有助于理解代謝轉(zhuǎn)變在纖維素酶生產(chǎn)中的影響。基因組重測序揭露了一些可能影響纖維素酶生產(chǎn)的新的位點和其他一些被忽視的領(lǐng)域。我們的研究為鑒定更多的涉及纖維素酶生產(chǎn)的基因提供了資源,這為構(gòu)建更高效的生產(chǎn)菌株提供了堅實的理論基礎(chǔ)。此外,我們也構(gòu)建了里氏木霉光控表達(dá)系統(tǒng)用于異源蛋白表達(dá)。2)里氏木霉的兩個質(zhì)膜H~+-ATP酶通過基因敲除策略被首次鑒定和功能化表征。通過分析我們發(fā)現(xiàn)基因tre76238作為質(zhì)膜H~+-ATP酶在里氏木霉中發(fā)揮主要功能,而基因tre78757發(fā)揮次要功能�；騮re78757的敲除并不影響菌株表型,而基因tre76238的敲除則會損害菌株將質(zhì)子從胞內(nèi)泵出到胞外的能力,pH穩(wěn)態(tài)的失調(diào)導(dǎo)致菌株在葡萄糖培養(yǎng)下能夠持續(xù)的累積纖維素酶。轉(zhuǎn)錄水平分析顯示在敲除菌株De1238中纖維素酶合成相關(guān)的基因的轉(zhuǎn)錄水平大幅度提升。盡管xyr1的轉(zhuǎn)錄水平并沒有提升,但是EMSA分析顯示在菌株De1238中的確有其他的蛋白與cbh1啟動子發(fā)生了結(jié)合。通過pull-down技術(shù)以及質(zhì)譜分析,三個可能涉及纖維素酶基因調(diào)控的鋅指蛋白被鑒定。這些發(fā)現(xiàn)為里氏木霉纖維素酶的表達(dá)調(diào)控提供了新的見解,同時也為通過調(diào)節(jié)胞內(nèi)pH穩(wěn)態(tài)來改善絲狀真菌纖維素酶的生產(chǎn)提供了新的策略。3)為了挖掘新的糖苷水解酶,我們利用棉花生物質(zhì)作為碳源對土壤樣品微生物進(jìn)行了富集培養(yǎng)。為了理解棉花生物質(zhì)的降解過程,我們對微生物群落分泌的糖苷水解酶譜進(jìn)行表征,結(jié)果顯示在這個微生物群落中細(xì)胞和纖維素底物之間的物理接觸是纖維素高效降解所必需的。通過16SrRNA分析,具有代表性的微生物群落結(jié)構(gòu)被鑒定,噬纖維細(xì)菌很可能在這個群落中對棉花生物質(zhì)降解起重要作用。通過對宏基因組序列的分析,32個主要的糖苷水解酶家族被鑒定,總共含有2058個候選的基因。16個糖苷水解酶編碼基因被克隆并在大腸桿菌中成功表達(dá),這些蛋白分別對4-硝基苯基-N-乙�；�-β-D-氨基葡糖苷、4-硝基苯基-β-D-木糖苷、昆布多糖、4-硝基苯基-β-D-葡萄糖苷、4-硝基苯基-β-D-葡糖苷酸、羧甲基纖維素和4-硝基苯基-β-D-甘露糖苷具有水解活性。此外,3個蛋白與最近似的同源物的一致性低于60%。土壤微生物基因組分析為挖掘新型生物質(zhì)降解酶提供了良好的策略�？寺〉氖畮讉�糖苷水解酶在生物質(zhì)轉(zhuǎn)化和產(chǎn)品生產(chǎn)中也具有潛在的應(yīng)用價值。我們的研究為理解植物生物質(zhì)降解的途徑以及酶的組成和相互作用提供了一定的見解。
[Abstract]:Trichoderma reesei is one of the most important producers of biodegradable enzymes. The enzymes produced by Trichoderma reesei are widely used in industrial production. Some high-yield mutant strains of Trichoderma reesei have been obtained by traditional mutagenesis methods. However, the high cost of producing biodegradable enzymes remains a great challenge for their commercial application. In Trichoderma reesei, pH is also an important factor affecting cellulase production. Plasma membrane H + - ATPase regulates intracellular pH homeostasis and nutrient uptake. However, the function of plasma membrane H~+-ATPase of Trichoderma reesei has not been studied so far. The combination of glycoside hydrolases has been widely used in industrial production. In order to meet the demand of glycoside hydrolases in industrial production, it is urgent to develop high-performance glycoside hydrolases to reduce the production of glycoside hydrolases. Macro-genomics has become a powerful method to study the diversity of microbial communities and explore new biocatalysts directly. In order to develop more efficient enzyme-producing strains and dig new glycoside-hydrolyzing enzymes, the following three aspects were studied in this paper. 1) Trichoderma reesei mutant strain SS-II Compared with strain RUT-C30, SS-II with intact Cre1 protein exhibited faster growth and about 1.5 times higher CMC activity in microcrystalline cellulose or lactose culture. On this basis, transcriptional data of 65 cellulose-degrading enzymes, 41 transcription factors and 152 transporters were further analyzed. To identify genetic mutations in S-II, the genome was sequenced. In S-II, 184 genes were sequenced. In addition, 157 genes affected by the mutation were identified. Most of these genes involved transport, secretion, protein metabolism and transcription. Nine genes affected by the mutation in S-II were further analyzed. Three of them were knocked out in RUT-C30 under microcrystalline cellulose culture. The comparative analysis of the transcriptomes of strains SS-II and RUT-C30 helps to understand the effect of metabolic transformation on cellulase production. Genome sequencing reveals new sites that may affect cellulase production and other neglected areas. Genes involved in cellulase production provide a solid theoretical basis for the construction of more efficient production strains. In addition, we have also constructed a Trichoderma reesei photocontrol expression system for heterologous protein expression. 2) Two plasmalemma H~+-ATPases of Trichoderma reesei have been identified and characterized for the first time by gene knockout strategy. We found that tre 76238 plays a major role as plasma membrane H~ + - ATPase in Trichoderma reesei, while tre 78757 plays a minor role. The knockout of TRE 78757 does not affect the phenotype of the strain, while the knockout of TRE 76238 impairs the ability of the strain to pump protons from intracellular to extracellular, and the imbalance of pH homeostasis leads to the strain's presence in Portuguese. Transcription level analysis showed that the transcription level of genes related to cellulase synthesis in the knockout strain De1238 increased significantly. Although the transcription level of xyr1 did not increase, EMSA analysis showed that other proteins in the strain De1238 did bind to the promoter of cbh1. Three zinc finger proteins that may be involved in the regulation of cellulase genes were identified by pull-down technique and mass spectrometry. These findings provide new insights into the regulation of cellulase expression in Trichoderma reesei, and provide new strategies for improving filamentous fungal cellulase production by regulating intracellular pH homeostasis. In order to understand the degradation process of cotton biomass, we characterized the glycoside hydrolase spectrum secreted by the microbial community. The results showed that there was physical contact between cells and cellulose substrates in the microbial community. Through 16S rRNA analysis, representative microbial community structures were identified, and cellophagous bacteria were likely to play an important role in the degradation of cotton biomass in the community. Gene. 16 glycoside hydrolase encoding genes were cloned and successfully expressed in E. coli. These proteins were 4-nitrophenyl-N-acetyl-beta-D-aminoglycoside, 4-nitrophenyl-beta-D-xylanoside, kumbu polysaccharide, 4-nitrophenyl-beta-D-glucoside, 4-nitrophenyl-beta-D-glucosidate, carboxymethyl cellulose and 4-nitrophenyl-beta-beta-xylanoside, respectively. D-mannoside has hydrolytic activity. In addition, the consistency of the three proteins with the nearest similar homologues is less than 60%. Soil microbial genome analysis provides a good strategy for the discovery of novel biomass degrading enzymes. Cloned dozens of glycoside hydrolases also have potential applications in biomass transformation and product production. It provides some insights into understanding the pathway of plant biomass degradation and the composition and interaction of enzymes.
【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】：2017
【分類號】：TQ925
，
本文編號：2207967
[Abstract]:Trichoderma reesei is one of the most important producers of biodegradable enzymes. The enzymes produced by Trichoderma reesei are widely used in industrial production. Some high-yield mutant strains of Trichoderma reesei have been obtained by traditional mutagenesis methods. However, the high cost of producing biodegradable enzymes remains a great challenge for their commercial application. In Trichoderma reesei, pH is also an important factor affecting cellulase production. Plasma membrane H + - ATPase regulates intracellular pH homeostasis and nutrient uptake. However, the function of plasma membrane H~+-ATPase of Trichoderma reesei has not been studied so far. The combination of glycoside hydrolases has been widely used in industrial production. In order to meet the demand of glycoside hydrolases in industrial production, it is urgent to develop high-performance glycoside hydrolases to reduce the production of glycoside hydrolases. Macro-genomics has become a powerful method to study the diversity of microbial communities and explore new biocatalysts directly. In order to develop more efficient enzyme-producing strains and dig new glycoside-hydrolyzing enzymes, the following three aspects were studied in this paper. 1) Trichoderma reesei mutant strain SS-II Compared with strain RUT-C30, SS-II with intact Cre1 protein exhibited faster growth and about 1.5 times higher CMC activity in microcrystalline cellulose or lactose culture. On this basis, transcriptional data of 65 cellulose-degrading enzymes, 41 transcription factors and 152 transporters were further analyzed. To identify genetic mutations in S-II, the genome was sequenced. In S-II, 184 genes were sequenced. In addition, 157 genes affected by the mutation were identified. Most of these genes involved transport, secretion, protein metabolism and transcription. Nine genes affected by the mutation in S-II were further analyzed. Three of them were knocked out in RUT-C30 under microcrystalline cellulose culture. The comparative analysis of the transcriptomes of strains SS-II and RUT-C30 helps to understand the effect of metabolic transformation on cellulase production. Genome sequencing reveals new sites that may affect cellulase production and other neglected areas. Genes involved in cellulase production provide a solid theoretical basis for the construction of more efficient production strains. In addition, we have also constructed a Trichoderma reesei photocontrol expression system for heterologous protein expression. 2) Two plasmalemma H~+-ATPases of Trichoderma reesei have been identified and characterized for the first time by gene knockout strategy. We found that tre 76238 plays a major role as plasma membrane H~ + - ATPase in Trichoderma reesei, while tre 78757 plays a minor role. The knockout of TRE 78757 does not affect the phenotype of the strain, while the knockout of TRE 76238 impairs the ability of the strain to pump protons from intracellular to extracellular, and the imbalance of pH homeostasis leads to the strain's presence in Portuguese. Transcription level analysis showed that the transcription level of genes related to cellulase synthesis in the knockout strain De1238 increased significantly. Although the transcription level of xyr1 did not increase, EMSA analysis showed that other proteins in the strain De1238 did bind to the promoter of cbh1. Three zinc finger proteins that may be involved in the regulation of cellulase genes were identified by pull-down technique and mass spectrometry. These findings provide new insights into the regulation of cellulase expression in Trichoderma reesei, and provide new strategies for improving filamentous fungal cellulase production by regulating intracellular pH homeostasis. In order to understand the degradation process of cotton biomass, we characterized the glycoside hydrolase spectrum secreted by the microbial community. The results showed that there was physical contact between cells and cellulose substrates in the microbial community. Through 16S rRNA analysis, representative microbial community structures were identified, and cellophagous bacteria were likely to play an important role in the degradation of cotton biomass in the community. Gene. 16 glycoside hydrolase encoding genes were cloned and successfully expressed in E. coli. These proteins were 4-nitrophenyl-N-acetyl-beta-D-aminoglycoside, 4-nitrophenyl-beta-D-xylanoside, kumbu polysaccharide, 4-nitrophenyl-beta-D-glucoside, 4-nitrophenyl-beta-D-glucosidate, carboxymethyl cellulose and 4-nitrophenyl-beta-beta-xylanoside, respectively. D-mannoside has hydrolytic activity. In addition, the consistency of the three proteins with the nearest similar homologues is less than 60%. Soil microbial genome analysis provides a good strategy for the discovery of novel biomass degrading enzymes. Cloned dozens of glycoside hydrolases also have potential applications in biomass transformation and product production. It provides some insights into understanding the pathway of plant biomass degradation and the composition and interaction of enzymes.
【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】：2017
【分類號】：TQ925
，
本文編號：2207967
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