里氏木霉纤维素酶高产菌株遗传改造及新型糖苷水解酶的挖掘
发布时间：2018-08-27 17:37
【摘要】：里氏木霉是生物质降解酶的重要生产者之一,其生产的酶被广泛应用于工业生产。一些里氏木霉高产突变菌株已经通过传统诱变方法获得。然而,生物质降解酶生产的高成本仍然是其商业化应用的巨大挑战。此外,这些突变菌株相应表型下的遗传机制仅仅被部分地理解。全面地理解遗传改变对纤维素酶生产的影响有利于开发更高效的纤维素酶生产菌株。在里氏木霉中,pH也是影响纤维素酶生产的重要因素。质膜H~+-ATP酶在调节胞内pH稳态和营养摄取等生理过程中发挥着关键作用。然而,里氏木霉的质膜H~+-ATP酶的功能到目前为止仍然没有被研究。多种糖苷水解酶的组合使用已经广泛应用于工业生产。为了满足糖苷水解酶在工业生产中的需求,工业上迫切需要具有高表现力的糖苷水解酶来降低生产成本。宏基因组学已经成为一种强有力的方法直接研究微生物群落的多样性和挖掘新型生物催化剂。为了开发更高效的酶制剂生产菌株和挖掘新的糖苷水解酶,本文从以下三个方面展开了研究。1)一株里氏木霉突变菌株SS-Ⅱ通过多次的NTG诱变在菌株NG14的基础上分离获得。与菌株RUT-C30相比,拥有完整Cre1蛋白的SS-Ⅱ在微晶纤维素或乳糖培养下展现出更快的生长和约1.5倍高的羧甲基纤维素酶活性。通过SS-Ⅱ和RUT-C30的转录组数据的对比分析,我们发现有1764个基因在微晶纤维素、葡萄糖、乳糖和小麦秸秆下差异化表达。在此基础上,65个纤维素降解相关的酶、41个转录因子和152个转运蛋白的转录数据被进一步分析。为了鉴定在SS-Ⅱ中发生的遗传突变,我们对其基因组进行了测序。在SS-Ⅱ中,总共有184个单核苷酸位点突变和40个小的插入/缺失被鉴定。此外,157个受突变影响的基因被鉴定。在这些基因中,大多数涉及运输、分泌、蛋白质代谢和转录。9个在SS-Ⅱ中受突变影响的基因被进一步分析。微晶纤维素培养下,在RUT-C30中敲除其中的3个基因会明显影响纤维素酶的生产。菌株SS-Ⅱ和RUT-C30的转录组比较分析有助于理解代谢转变在纤维素酶生产中的影响。基因组重测序揭露了一些可能影响纤维素酶生产的新的位点和其他一些被忽视的领域。我们的研究为鉴定更多的涉及纤维素酶生产的基因提供了资源,这为构建更高效的生产菌株提供了坚实的理论基础。此外,我们也构建了里氏木霉光控表达系统用于异源蛋白表达。2)里氏木霉的两个质膜H~+-ATP酶通过基因敲除策略被首次鉴定和功能化表征。通过分析我们发现基因tre76238作为质膜H~+-ATP酶在里氏木霉中发挥主要功能,而基因tre78757发挥次要功能。基因tre78757的敲除并不影响菌株表型,而基因tre76238的敲除则会损害菌株将质子从胞内泵出到胞外的能力,pH稳态的失调导致菌株在葡萄糖培养下能够持续的累积纤维素酶。转录水平分析显示在敲除菌株De1238中纤维素酶合成相关的基因的转录水平大幅度提升。尽管xyr1的转录水平并没有提升,但是EMSA分析显示在菌株De1238中的确有其他的蛋白与cbh1启动子发生了结合。通过pull-down技术以及质谱分析,三个可能涉及纤维素酶基因调控的锌指蛋白被鉴定。这些发现为里氏木霉纤维素酶的表达调控提供了新的见解,同时也为通过调节胞内pH稳态来改善丝状真菌纤维素酶的生产提供了新的策略。3)为了挖掘新的糖苷水解酶,我们利用棉花生物质作为碳源对土壤样品微生物进行了富集培养。为了理解棉花生物质的降解过程,我们对微生物群落分泌的糖苷水解酶谱进行表征,结果显示在这个微生物群落中细胞和纤维素底物之间的物理接触是纤维素高效降解所必需的。通过16SrRNA分析,具有代表性的微生物群落结构被鉴定,噬纤维细菌很可能在这个群落中对棉花生物质降解起重要作用。通过对宏基因组序列的分析,32个主要的糖苷水解酶家族被鉴定,总共含有2058个候选的基因。16个糖苷水解酶编码基因被克隆并在大肠杆菌中成功表达,这些蛋白分别对4-硝基苯基-N-乙酰基-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、羧甲基纤维素和4-硝基苯基-β-D-甘露糖苷具有水解活性。此外,3个蛋白与最近似的同源物的一致性低于60%。土壤微生物基因组分析为挖掘新型生物质降解酶提供了良好的策略。克隆的十几个糖苷水解酶在生物质转化和产品生产中也具有潜在的应用价值。我们的研究为理解植物生物质降解的途径以及酶的组成和相互作用提供了一定的见解。
[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.
【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】：TQ925
，
本文编号：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.
【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】：TQ925
，
本文编号：2207967
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