黑曲霉高效表达异源耐热β-葡萄糖苷酶分子策略和代谢机制研究

One of the biggest concerns of the twenty-first century is climate change resulting from burning fossil fuels. This fact makes the utilization of bioethanol as an attractive option for combating both global warming and consumption of crude oil. Further increase in bioethanol production may come from lignocellulose,which is abundant, inexpensive, and renewable in waste products. For bioethanol production, β-glucosidase plays a critical role in cellulose degradation. As a filamentous fungi，Aspergillus niger has the ability to produce a plethora of bioactive metabolites and enzymes enabling them to thrive in competitive environments. Well-tested fermentation processes and generally regarded as safe status make it an attractive protein production host. High throughput heterologous expression in an appropriate host constitute a potential bottleneck in functional genomics studies. The overproduction strategies of heterologous gene expression will increase the productivity of the given protein, such as using strong promoter, increasing gene dosage, controlling the proper folding of the protein within the undoplasmic reticulum, getting a correct protelytic cleavage of preprotein secretion, or reducing the fungal endogenous protease activity. To facilitate the use of filamentous fungi in functional genomics, we will develop various components namely promoters, markers, reporters and a suitable host background to synthesize the expression cassette and present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus niger as protein production platforms,which allows rapid and easy heterologous expression of a gene of interest. These results will reveal the assembly and secretion route of heterologous protein, deepen and enrich content of heterologous expression theory in filamentous fungi.

化石能源消耗导致的气候变化是本世纪最令人关注的问题之一。作为替代能源，生物质能源成为研究热点。纤维素是自然界中利用潜力最大的可再生资源，β-葡萄糖苷酶在其降解生产酒精过程中起关键作用。针对黑曲霉难以转化、异源蛋白合成效率低、易被异源酸性蛋白酶分解、转化子异源基因遗传稳定性差的瓶颈性问题，利用DNA介导转化系统引入异源耐热β-葡萄糖苷酶基因组，在产β-葡萄糖苷酶活性高的胞外蛋白酶缺失株中表达，规避已合成异源蛋白在胞内被改性和分泌后受降解的风险，以期获得产酶活性更高的突变株，筛选高产β-葡萄糖苷酶的菌株。同时，应用各种基因工程方法构建融合表达体系来提高稳定性，采用强启动子控制、引入多拷贝基因、表达融合蛋白、缺失蛋白酶活力、信号肽修饰等提高重组蛋白表达的技术策略，进行表达盒全合成并构建高效表达载体，以期建立黑曲霉异源蛋白的高效表达平台，揭示合成与分泌途径，丰富丝状真菌表达异源蛋白的方法和理论。

纤维素酶高效分解植物纤维原料产糖成为国际研究的热点，但产酶菌株活性不高是纤维素酶生产和应用中的瓶颈难题。β-葡萄糖苷酶是纤维素酶的限速酶，因此通过基因重组技术构建高活性β-葡萄糖苷酶对纤维素有效降解具有重要意义。本项目首先采用超滤、亲和层析、凝胶过滤层析等技术对耐热β-葡萄糖苷酶进行了分离纯化，研究了其主要酶动力学特性，基因克隆及序列分析结果表明酶蛋白含860个氨基酸残基，存在4个疏水区，二级结构含α-螺旋和β-折叠，结构域具有(β/α)8 TIM桶域、(β/α)6夹层三明治域和纤维连接蛋白的III-like域。进而构建了酶强表达载体pCAMBIA1301-GlaA promoter-3.316bgl1-3.2130Ter和pCAMBIA1301-3.2130 promoter-3.316bgl1-3.2130Ter。通过农杆菌介导转化将异源耐热酶基因组在产β-葡萄糖苷酶活性高的胞外蛋白酶缺失菌株中进行了表达，采用强启动子控制、引入多拷贝基因、表达融合蛋白、缺失蛋白酶活力等技术策略提高了稳定性。对酶基因序列同源性分析结果显示重组菌株和黑曲霉β-葡萄糖苷酶同源率高达95.53%。此外对异源耐热β-葡萄糖苷酶合成培养条件进行了研究，建立了重组黑曲霉菌体生长动力学模型、酶合成动力学模型及底物消耗动力学模型。通过生物学分析显示黑曲霉表达系统中蛋白代谢通路为蛋白折叠模块，囊泡运输模块，内质网相关降解模块和胁迫应激反应模块，各个功能之间相互影响。在项目支持下，建立了黑曲霉异源活性蛋白的高效表达体系，揭示了异源蛋白合成与分泌途径，丰富了丝状真菌表达异源蛋白的技术方法和基本理论，为β-葡萄糖苷酶在生物质能源中的应用提供理论指导。