酿酒酵母中Cwp2p和BGLI表面展示高表达下的蛋白质量控制响应及其调控研究

Surface display is an efficient method to produce the whole cell biocatalyst. The inadequacy on its fundamental principle research restricted to further improve the productivity of displayed enzyme and apply it into industrial production process. Saccharomyces cerevisiae is an excellent model organism for protein trafficking study. In our previous study, it was found to be difficult to further increase the displayed expression level of heterogenous β-glucosidase BGLI by using the anchor of S. cerevisiae cell wall protein Cwp2p and extracellular secretion still existed. So the possible protein quality control mechanism behind this phenomenon would be analyzed. In this study, Cwp2p, a typical and major constituent of the cell wall, and BGLI-Cwp2p anchor are selected as targets to investigate the complex cell responses to the increasingly expressed or even over-expressed level, which would center on UPR/ERAD pathway, GPI protein trafficking control and yapsin protease. The genetic, biochemical and omics methods, as well as real-time and dynamic targeting analysis by GFP marker will be used together. The quality control systems specific for Cwp2p and BGLI-Cwp2p anchor would be identified and the interaction between them would be studied. On this base, the limiting factors to obtain the highly anchored, abundant and host-compatible surface-displaying expression of heterologous protein would be investigated by tuning the expression level of the key and important genes in quality control systems. The results of this study will help to develop the fundamental theory of protein trafficking and the yeast surface-display technology.

表面展示是制备全细胞催化剂的有效手段，但相关基础研究的缺乏制约了展示酶产率提高和大规律制造工艺的应用。本研究以前期利用Cwp2p锚定区anchor进行外源BGLI表面展示中表达水平难以进一步提高、少量胞外分泌的现象为切入点，分析展示蛋白大量合成、内质网压力升高下的质量控制系统反应。研究以Cwp2p和BGLI-Cwp2p anchor为对象，结合使用遗传、生化、组学手段和基于GFP标记的实时动态定位分析，考察随Cwp2p和BGLI-Cwp2p anchor表达水平上升直至过量时以UPR/ERAD、GPI蛋白转运和yapsin蛋白酶响应为核心的综合效应，鉴定GPI蛋白特异性质量控制机制，揭示这些机制之间相互关系；通过这些质量控制系统中关键基因的表达调控，考察表面展示达到高水平表达、高锚定效率、高宿主兼容性的制约因素及其实现措施。本研究成果有助于丰富蛋白定位基础理论，为表面展示提供技术支撑。

表面展示是制备全细胞催化剂的有效手段，而酿酒酵母是极具应用价值的模式生物。本项目考察涉及更复杂调控机制的利用GPI锚定域进行BGLI表面表达的蛋白质量控制系统反应，以期实现更有效的表达。.首先考察和评价表达体系诸要素及其与宿主的兼容性。菌株W303-1A中优选了cwp2 anchor锚定表达盒；不同拷贝数下的锚定表达都表现出较分泌表达明显得多的低酶活和生长抑制，而扣囊复膜孢酵母来源BGLI表达菌株又较棘孢曲霉来源BGLI（AaBGLI）表达菌株为甚，其以YEplac195为载体锚定表达菌株最终生长被完全抑制。以产醇能力和抗逆性强的工业菌株An-α作为宿主，总酶活和细胞酶活比例均低于W303-1A对应值，但生长均没有被完全抑制。而内切葡聚糖酶表达的细胞酶活比例最高仅50%左右。.对An-α和W303-1A建立了循环使用URA3、基于δ序列同源臂的多拷贝整合技术和基于CRISPR-Cas9系统的高效整合方法；使用BGLI和内切葡聚糖酶进行了效果考察。构建了以UPRE为启动子、分别以LacZ、GFP和mCherry为报告基因的UPR指示菌株，考察了使用效果和对衣霉素、DTT及乙酸和乙醇等作为全胞催化剂可能涉及效应物的UPR响应谱。.在W303-1A(LEU2::UPRE-LacZ)和An-α(LEU2::UPRE-LacZ)中表达AaBGLI，拷贝数梯度通过染色体上不同拷贝数整合和以YCplac33或YEplac195为载体导入相结合实现。LacZ酶活测定和RT-PCR分析显示了BGLI酶活水平与UPR响应值和HAC1i水平亦即内质网胁迫压力间良好的正相关性；在分泌表达对应值始终高于同样拷贝数下锚定表达对应值的情况下，W303-1A对应值始终高于An-α对应值。锚定表达菌株的细胞酶活占总酶活的比例基本不受拷贝数影响。转录水平分析显示了锚定表达菌株GPI加工和转运相关关键基因表达普遍高于分泌表达菌株，进一步的分析推测锚定表达总量首要制约因素在ER。.宿主染色体上基因修饰结果显示：Yps1p和Yps2p基因缺陷没有显著影响锚定效率和总酶活，锚定表达中的胞外分泌与Yps1p和Yps2p基因的关系还待进一步研究；而过表达Pdi1p和Kar2p能提高BGLI锚定表达总酶活；过表达p24复合物组分Erv25能提高细胞表面酶活。.研究有助于丰富蛋白定位基础理论，为表面展示提供技术支撑。