基于无机氮源信号开关的适时自诱导型曲霉表达体系的构建及优化

The filamentous fungi Aspergilli have great potential for heterologous protein production due to their powerful protein posttranslational modification and secretion abilities, nutritional flexibility and metabolic capacities. However, the prevalent fungal hosts including A. oryzae and A. niger, have bottleneck such as low recombination efficiencies, too long growth cycles, limited production levels, and especially lack of controllable expression modes. Our previous study (Zhou SM, Nature Chem Biol.2013) and other related researches revealed the following nitrogen metabolism regulation and growth features of A. nidulans: The transcription of nitrogen assimilatory genes are induced by NO3- and repressed by NH4+. Additionally, NH4+ is advantage nitrogen source rather than NO3- for cell growth; A. nidulans is genetically well-characterized fungus with fast growth, high recombination efficiency and abundant genetic engineering tools. In this project, based on the accumulated knowledge, we will isolate and employ a NO3-/NH4+-response promoter to create a novel A. nidulans expression system which is strictly controlled by the rate of NO3-/NH4+ in medium. Since the advantage nitrogen source NH4+ is used for both cell growth and expression repression, and NO3- is used as expression inducer, the stage of cell growth and protein expression can be separated spontaneously. In order to further improve the expression level and lower the background expression level, several new genetic engineering strategies will be developed and integrated into the host cells using CRISPR/cas9. The nitrogen source based and auto-regulatory fungus expression system combines the advantages of both constitutive and inducible expression systems, and provide a novel mode for expression systems.

蛋白表达系统是生命科学研究的必要工具。曲霉兼具蛋白翻译后修饰和易操作优点，有望成为继大肠杆菌、酵母和动物细胞后更加理想的表达宿主。但现有曲霉因基因重组效率低和缺乏理想的蛋白表达调控手段，限制了其广泛应用。我们的前期工作（Zhou SM, Nature Chem Biol. 2013）及相关研究发现构巢曲霉生长快、重组效率高，生长代谢所需氮同化基因受NO3-诱导和NH4+抑制正负调控, 且NH4+的利用优先于NO3-。本课题基于前期发现，设计并优化受NO3-/NH4+正负调控的启动子，建立适时响应NO3-/NH4+信号的表达载体，使曲霉在对数生长中期自动诱导外源蛋白表达，实现菌体生长和蛋白表达适时分离；进而定向改造宿主增强表达活性及调控严密性；最终通过CRISPR技术将表达载体整合入宿主基因组，创建基于NO3-/NH4+信号介导的适时自诱导型曲霉表达体系，推动新型蛋白表达系统的建立与发展。

曲霉有潜力成为优良的外源蛋白表达宿主，但现有的表达模式启动子种类有限，且调控严密性欠佳。本课题开发以构巢曲霉为表达宿主，以不同氮源为诱导剂和抑制剂实现霉菌自诱导表达。. 研究内容包括表征氮源同化基因NiaD，NiiA和NrtA的启动子性质，筛选出受NO3-强力诱导和NH4+严密抑制的启动子。将该启动子作为曲霉外源蛋白表达用启动子筛选出能自动将菌体生长和蛋白生产分开的培养条件，实现曲霉表达的自诱导。另外，优化启动子进一步提高表达效率。构建了以niaD.P和prxA.P串联型蛋白表达体系，研究是否可以将niaD.P诱导的严密性和prxA.P的高强度融合为更高效的表达体系。最后基于氮源调控的严密性，尝试将niaD.P用于敲除致死型基因的功能性研究。. 本研究在氮源同化过程中多个基因启动子的表征，及在霉菌蛋白表达体系构建和霉菌致死型基因的功能研究方面均取得进展。. 启动子表征方面：确定启动子niaD.P和nrtA.P适合曲霉表达体系。发现nrtA.P在硝酸盐和氮源饥饿条件下被激活，在其他氮源条件下被抑制。nrtA.P活性受AreA和nirA调控，AreA调控作用发生在硝酸盐和氮源饥饿条件下，而NirA的调控作用仅发生在硝酸盐条件下。另外发现了启动子中重要的转录因子四个结合位点。. 霉菌蛋白表达体系方面：1，证实了niaD.P可用于外源性蛋白的表达；当氮源比例为80 mM NO3-/20 mM NH4+时可实现了曲霉的自诱导型表达体系。使用该表达体系成功表达了毒性蛋白。1，证实了niaD.P可用于外源性蛋白catalase的表达；当氮源比例为80 mM NO3-/20 mM NH4+时可实现了曲霉的自诱导型表达体系。使用该表达体系在含有5’-FOA试剂的培养基内表达pyrG基因，证实了该体系可用于对表达毒性的蛋白。2，通过nrtA.P启动子优化，活性提高了两倍。3，在将氮源同化启动子与响应H2O2的启动子prxA.P联合使用中发现，联用的曲霉表达体系比单独使用prxA.P的表达体系效率更好。。. niaD.P在霉菌必需基因的功能研究中：证实G6PD为必须基因，并发现霉菌可能通过调控G6PD的活性来实现NADPH的足量供应与prxA表达水平之间的平衡。