CAS OpenIR  > 研究所(批量导入)
里氏木霉纤维素酶成熟分泌过程限制因子的研究
王国坤
学位类型博士
导师陈树林
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词里氏木霉 纤维素酶 未折叠蛋白响应 二硫键异构酶 硫氧还蛋白
摘要

纤维素酶是一种重要的工业酶,在纤维素糖化、造纸、纺织、酿酒等工业过程中广泛应用。里氏木霉是科学研究及工业生产中应用最为广泛的纤维素酶产生菌,里氏木霉纤维素酶分泌过程是在诱导条件下进行的,其分泌过程起始于菌体对环境信号的感知,细胞内的信号转导,进而发生纤维素酶编码基因的转录,经过核糖体的翻译,初生纤维素酶蛋白在内质网及高尔基体进行折叠和修饰,最终通过膜泡运输分泌到细胞外。通过基因工程进行菌种的理性设计是提高纤维素酶产量的有效手段,然而大量实验表明仅通过纤维素酶或其基因的改造,只能有限地提高纤维素酶的生产能力,并不满足实际生产的需要。而深度解析里氏木霉纤维素酶的调控、合成以及分泌机制,功能鉴定关键调控因子,可以为菌株理性设计提供改造靶点。本论文主要研究了里氏木霉中纤维素酶合成分泌过程中蛋白翻译后过程的不同途径的响应机制,以及阐明了参与蛋白质折叠过程中二硫键形成及打开的两个硫氧还蛋白家族成员:TrPDI2及TrTRX1对于里氏木霉生长及纤维素酶产生的作用,并分析其作用机理,进而揭示了环境胁迫对里氏木霉纤维素酶产生的影响。具体研究内容如下:1、以QM9414和Rut C30两个产酶能力不同的里氏木霉突变体为研究对象,在纤维素酶诱导条件下,对参与未折叠蛋白响应(UPR)、内质网降解途径及膜泡运输等各途径的潜在调控基因进行了转录水平分析。结果显示在以微晶纤维素为诱导物进行诱导的过程中,位于内质网的未折叠蛋白感受器、分子伴侣及折叠酶等的编码基因上调表达,而参与胞内膜泡转运环节的蛋白表达量无明显变化,因此推测以微晶纤维素为诱导物的诱导过程可以诱发UPR,而且相对QM9414菌株,Rut C30菌株中参与UPR的各种元件的表达更早且强度更高,同时胞内纤维素酶的存留量更少,而胞外蛋白质产量及纤维素酶活力更高。研究结果表明及时有力的未折叠蛋白响应,以及增强的胞内蛋白质转运可以提高里氏木霉纤维素酶的生产能力,因此提高里氏木霉蛋白折叠和修饰能力可能是一种可行的里氏木霉纤维素酶生产的改造策略。2、通过同源比对分析了里氏木霉中的二硫键异构酶(PDI)蛋白TrPDI2,其与黑曲霉TIGA、里氏木霉TrPDI1的序列同源性为36.94 和11.81 %;进一步通过失活的RNase的酶活性复性分析证实其具备二硫键异构酶活性;Trpdi2基因的过量表达显著提高了纤维素酶主要组分CBH1的分泌能力及活性。体外DTT处理CBH1蛋白及蛋白活性测定结果显示二硫键对于纤维素外切酶活性非常重要,因此推测Trpdi2通过提高二硫键形成能力促进纤维素外切酶的产生;另外免疫分析证实TrPDI2的表达水平及氧化还原状态都会响应纤维素酶的诱导,以提供持续的氧化力保证纤维素外切酶折叠。综上所述,TrPDI2作为存在于里氏木霉的二硫键异构酶,在纤维素酶诱导过程中,通过其表达水平提高以及氧化还原状态变换提高二硫键形成能力,因而促进蛋白质的折叠成熟,提高纤维素外切酶的分泌。3、以线粒体硫氧还蛋白(Trx)的编码基因Trtrx1为研究对象,解析里氏木霉的线粒体Trx在抗氧化胁迫下的作用机制。首先通过亚细胞定位及活性检测,证实TrTRX1是一种线粒体定位的硫氧还蛋白。在ΔTrtrx1突变体中,Trtrx1基因的缺失并不改变里氏木霉的生长,也未明显影响里氏木霉对于活性氧的抗性;在正常生长条件下缺失Trtrx1同样没有影响里氏木霉总GSH含量,但是在氧化胁迫下总GSH含量显著增加;另外外源GSH的添加引起Trx相关基因的表达量显著下降。因此推测GSH系统是里氏木霉线粒体中维持氧化还原状态的主导因素,而里氏木霉的线粒体硫氧还蛋白TrTRX1,不是里氏木霉正常生长及氧化胁迫抗性所必需的,有可能辅助性参与里氏木霉对氧化胁迫的抗性。本论文研究结果有助于深入理解里氏木霉纤维素酶合成分泌蛋白翻译后加工调控机制,加深对里氏木霉胁迫抗性系统的认识,为通过分泌过程及氧化胁迫抗性的改造提高产酶能力提供基础。

其他摘要

As an important industrial enzyme, cellulase finds applications in many industrial processes including cellulosic saccharification pulp textile and brewing. The Sordariomycete Trichoderma reesei (T. reesei, teleomorph Hypocrea jecorina) is a filamentous fungi cellulase producer that is most widely used for scientific research and industrial cellulase production. Cellulase produced by T. reesei is extracellular-targeted and inducible enzyme, the whole production procedure consist of sensing of inducer or signal, intracellular signaling to activate cellulase genes’ transcription, translation, protein folding and modification of primary peptide in endoplasmic membrane and Golgi apparatus, secretion of correctly folded protein to extracellular in vesicle-mediated transport. Rational strain improvement was attempted, however, manipulations of cellulases and their encoding genes failed to achieve the enzyme-producing level comparable to industrial application. Understanding the mechanism of cellulase production, regulation and secretion would provide more targets for strain improvement. Also, screening of key limiting factors is important for target identification. In this study, the effects on cellulase production by T. reesei of secretion and environmental stress was assessed, the roles of TrPDI2 and TrTRX1, two thioredoxin family members involved in disulfide bond formation and breakup were investigated, in growth and cellulase production in T. reesei, and the underlying mechanisms were analyzed.The effects of modification and secretion upon cellulase production are still unclear. To answer this question, transcript levels of cellulases, UPR elements and secretion-involved components were analyzed, and the differences between QM9414 and Rut C30, two T. reesei mutants with different cellulase-producing capability, were compared during cellulase induction by cellulose. Cellulase induction by cellulose drastically up-regulated expressions of the sensor of unfolded protein, chaperone and folding-assisted enzymes in endoplasmic reticulum, and resulted in UPR and low-grade increase in secretory transporters’ expression similar to that of chemical treatment. Rut C30 demonstrated earlier and more sustainable expressions of elements involved in UPR and lower amount of cellular retained cellulase compared to QM9414, indicating that Rut C30 had hypercellulolytic property partially for its earlier and enhanced UPR to more efficiently dispose of protein. Thus, the improvement in acceleration of protein folding and modification after translation may be a feasible approach for rational strain improvement for cellulase production beside the enhancement of cellulases’ expression levels.Efforts were made in this study to characterize a PDI homolog, TrPDI2 in T. reesei which exhibited a 36.94 % and an 11.81 % similarity to A. niger TIGA and T. reesei TrPDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.This study identified a Trx-encoding gene, Trtrx1 by subcellular localization and activity measurement. Loss of Trtrx1 however, did not lead to either significant morphology abnormality under normal and oxidative stress condition, or detectable difference in reactive oxygen species resistance. The unchanged GSH amount in Trtrx1 deletion strain under normal condition and slight increase under oxidative stress condition, as well as the interplay between Trx and GSH systems suggested that GSH system was dominant and sufficient to maintain the mitochondrial redox state in T. reesei, where TrTRX1 played a role as the backup oxidative stress resistance. Hence Trtrx1 was speculated to be a dispensable mitochondrial Trx in T. reesei during normal and oxidative stress condition but may play underlying role in the backup oxidative stress resistance and apoptosis in T. reesei.These results were helpful for understanding the roles of secretion in cellulases and the stress resistance system of T. reesei, providing information for improving cellulase-producing capability by manipulation of secretion pathway and redox defense system. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22875
专题研究所(批量导入)
推荐引用方式
GB/T 7714
王国坤. 里氏木霉纤维素酶成熟分泌过程限制因子的研究[D]. 北京. 中国科学院研究生院,2016.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可
里氏木霉纤维素酶成熟分泌过程限制因子的研(4149KB)学位论文 开放获取CC BY-NC-SA请求全文
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[王国坤]的文章
百度学术
百度学术中相似的文章
[王国坤]的文章
必应学术
必应学术中相似的文章
[王国坤]的文章
相关权益政策
暂无数据
收藏/分享
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。