产耐热β-葡萄糖苷酶黑曲霉对高温胁迫的生理响应及其调节机制的蛋白质组学研究

Fibre is the main raw material for producing ethanol- the second generation fuel. Efficient enzyme hydrolysis is always the research hotspot.Aspergillus niger has potential industrial application to produce β-glucosidase, which is one of the important part of cellulases.Heat shock is one of the environmental stresses that industrial microbes of all types will experience. The study of these environmental stress responses in microbes will illuminate many of the features now viewed as central to our understanding of cell regulating mechanisms. Transcriptional and Proteomics activation plays an important role in driving the multifaceted reaction to elevated temperature. This study characterizes the regulation of components of the heat shock response, a cellular stress defense mechanism present in Aspergillus niger. The hallmark of the heat shock response is an increase in heat shock protein expression, which results from transcriptional activation accomplished by heat shock transcription factors. This study will provide the new advances to the development of whole genome analyses led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, progress in the understanding of induction of the heat stress response will be summarized here. Although these stress conditions represent ancient challenges to Aspergillus niger and other filamentous fungi, much remains to will be learned about the mechanisms dedicated to dealing with these environmental parameters. Here the results of proteomic regulatory mechanism for resistance and sensitivity to a sudden heat shock and the identification of functions that are critical for genetic engineering breeding the excellent industrial strain.

纤维素是第二代燃料乙醇的主要原料，高效酶解一直是研究热点。β-葡萄糖苷酶是纤维素高效分解的关键酶。黑曲霉是合成β-葡萄糖苷酶最具工业应用前景的生产菌株。高温协迫是工业微生物发酵经常经历的环境协迫之一，阐明抗逆响应的分子调节机制，对于基因工程选育耐热工业生产菌种具有重要意义。为了构建耐高温黑曲霉基因工程菌株的提供理论依据，明确黑曲霉的耐热机理，本研究拟在蛋白质组学层面分析黑曲霉高温胁迫前后胞内外蛋白质动态变化情况，采用基因工程方法构建应激蛋白缺陷菌株证实应激蛋白的作用，对黑曲霉产β-葡萄糖苷酶过程的热激效应生理响应和调节机制进行分析，从转录组学层面进行全基因组分析相关基因表达重组和调节的信号网络途径，揭示丝状真菌对环境协迫的抗逆机制。本研究结果将进一步揭示丝状真菌环境协迫响应机制，丰富应激蛋白分子调控理论，为选育耐高糖、高温、高代谢物等逆境条件的生产菌株，提高工业生产效率提供技术和理论支撑。

黑曲霉具有生长速度快、营养要求简单、发酵成本低以及便于大规模工业化培养等优点，是合成纤维素酶系重要酶-β-葡萄糖苷酶的最具工业应用前景的生产菌株之一。高温耐热菌在实际生产中具有重要意义，洞悉耐热机理是构建耐高温基因工程菌株的基础。在基金的支持下，项目组开展了以下研究工作：首先，对耐热β-葡萄糖苷酶活性位点进行了分子动力学模拟研究，通过对比发现耐热β-葡萄糖苷酶二级结构中20.58% 的α-螺旋增加了蛋白的耐热性；其半胱氨酸和组氨酸含量较低，谷氨酸、天冬氨酸、精氨酸、异亮氨酸、赖氨酸氨基酸残基含量较高，蛋白中氨基酸的组成和结构在一定程度上提高了β-葡萄糖苷酶的热稳定性；模拟分析发现G510残基对β-葡糖苷酶的热稳定性影响最大，设计了3 个β-葡糖苷酶耐热突变体为定向改造酶的热稳定性提供了依据，同时为理解酶蛋白的热稳定机制提供了参考。其次，研究了菌体热胁迫耐受性与超微结构变化，观察了细胞生理形态改变，菌体长度随着温度升高而发生的显著变化，热胁迫菌体生理活动发生紊乱且造成了不良发育；黑曲霉细胞内通过改变其脂肪酸、磷脂和麦角固醇的组成来适应温度的变化，进而改变细胞膜流动性；热胁迫处理造成黑曲霉细胞海藻糖分泌量显著上调，以此来对抗外界胁迫能力。最后，对黑曲霉 3.316 进行了全基因组测序及分析，通过 iTRAQ标记技术联合液相二级质谱技术分析，建立了基因组学和蛋白组学的关系，发现高温胁迫前后细胞内外蛋白质组共有1025种蛋白质出现差异表达，黑曲霉热激分子伴侣Hspssc1、Hsp70伴侣和Hsp98 / Hsp104 / ClpA表达量明显上升；对高温胁迫前后差异表达蛋白进行了生物学分析，同时结合高温胁迫前后细胞膜的流动性、细胞膜成分改变情况以及胞内多糖含量变化情况，探究了黑曲霉对高温胁迫的耐受机理。