马克斯克鲁维酵母的未折叠蛋白响应

Kluyveromyces marxianus （KM）is considered as a kind of safe (GRAS) yeast species, emerging as a promising host for expressing proteins used for food, medicine and feed. Key to industrial application of KM expression system is to improve its secretion of exogenous proteins. Unfolded protein response (UPR) copes with stresses arisen from accumulation of misfolded or unfolded proteins in the lumen of endoplasmic reticulum in eukaryotic cells. UPR exists as a signaling network composed by stress sensors and downstream genes, which contributes to secretion of proteins. Through mutagenesis, we obtained a KM strain displaying high-level secretion of exogenous proteins. The transcriptional level of several UPR genes, as well as the resistance to a UPR inducer was increased in this strain, suggesting strengthened capacity of UPR underlies the improvement of secretory expression in KM. To explore UPR in KM, this project will perform following studies: 1) identification of sensors of UPR in KM by CRISPR; 2) identification of genes subjected to the regulation of UPR by RNA-seq; 3) identification of mutations that affect UPR in the KM strain displaying high-level secretory expression；4) development of strategies to improve secretory expression of KM by rational modification of UPR. 5) identification of mechanism by which novel UPR genes regulates secretory expression. This project will provide a better understanding of UPR network, and contribute to the industrial application of KM expression system.

马克斯克鲁维酵母(KM)是新兴的食品级表达宿主菌，生产食、药、饲用蛋白的潜力巨大。提升KM的分泌表达能力是加速其产业化的基础。未折叠蛋白响应（UPR）是真核细胞处理蛋白异常折叠胁迫的信号通路网络，由感应蛋白及其操纵的众多基因组成，它保障了蛋白的正常分泌。申请人团队通过诱变获得高效分泌外源蛋白的KM菌株。该菌株中多个UPR基因的转录水平上调，且对UPR诱导剂的抗性提高，提示增强UPR能提升KM的分泌表达能力。针对KM中UPR研究的空白，本课题将：1)利用CRISPR改造，鉴定KM中UPR的感应蛋白；2)采用RNA-seq，鉴定受UPR操纵的基因；3)鉴定高分泌KM菌株中影响UPR的突变； 4)通过理性改造UPR来促进KM的分泌表达；5)鉴定新型UPR基因影响蛋白质分泌表达的机制。本课题将为深入了解UPR网络提供重要信息，为促进KM系统的工业应用奠定基础。

马克斯克鲁维酵母是新兴的蛋白质表达宿主菌，提升马克斯克鲁维酵母的分泌表达能力是加速其产业化的基础。未折叠蛋白响应（UPR）是真核细胞处理蛋白异常折叠胁迫的信号通路网络。本项目发现马克斯克鲁维酵母拥有Bip-Ire1-Hac1的经典通路。ire1和hac1突变体对于UPR诱导剂DTT和衣霉素敏感。在DTT和衣霉素的刺激下，GRP78(编码Bip同源蛋白)的转录水平快速上升。此外还有114个基因的转录水平明显上调，包括了20个没有功能注释的新基因。我们利用过量表达的方式分析了97个UPR相关基因对于不同外源蛋白的表达水平的影响。24个基因的过量表达会明显提升基础表达水平较高的阿魏酸酯酶AnFaeA的分泌表达，其中编码丙酮酸脱羧酶的PDC1的过量表达可将表达水平提升2倍。有4个基因的过量表达，会明显抑制AnFaeA的分泌表达。有35个基因的过量表达会明显提升基础表达水平较低的内切葡聚糖酶RuCelA的表达。其中伴侣蛋白Caj1和GID复合物部分亚基的过量表达可以同时促进两种蛋白的分泌表达。研究结果为深入了解UPR网络的作用机制，并为推动马克斯克鲁维酵母的工业化应用奠定了重要的基础。.为了获得强启动子对UPR相关基因进行过量表达，本项目针对马克斯克鲁维酵母的菊粉酶启动子的活性开展了研究。本研究发现菊粉酶启动子中的InuR转录因子的一个潜在结合位点的突变，可以将外源基因的转录和表达水平提升2倍以上。利用该启动子，可在发酵罐级别实现木质纤维素降解酶的高效表达。该成果为马克斯克鲁维酵母表达系统的工业化应用提供了重要的表达元件。