以甘油受限誘導策略誘導Mxr1再程序化之嗜甲醇酵母菌生產木聚醣酶

Abstract

Pichia pastoris是嗜甲醇酵母菌的一種，為極具潛力的異源蛋白質表達系統，其優勢在於培養成本低、生長快速和產量高等，同時具有原核表達系統不具備的轉譯後修飾，因此廣泛應用生產重組蛋白質。然而AOX1啟動子 (alcohol oxidase I promoter) 調控嚴謹，使P. pastoris只能使用有毒、易燃的甲醇作為碳源時，才可誘導AOX1啟動子啟動，產出重組蛋白質。本研究希望發展非甲醇的誘導方式，來生產重組蛋白質，以改善甲醇誘導所帶來的缺點。本研究延續實驗室前人的研究，以額外表現MXR1之菌株進行實驗，並搭配AOX1啟動子表現木聚醣酶，結果確認額外表現MXR1菌株可在甲醇誘導時，提升重組蛋白質產量。以甘油受限誘導的方法進行無甲醇製程研究，額外表現MXR1菌株在醱酵槽的培養可成功誘導木聚醣酶的表現。以轉錄因子MXR1、甘油運輸蛋白GT1 (glycerol transporter I) 之基因表現量可以解釋甘油受限時可誘導目標蛋白質表達的原因；並以氨甲醯磷酸合成酶CPA1 (carbamoyl phosphate synthetase I) 之基因表現量解釋，在甘油受限下，缺乏碳源可由提高CPA1表現量，回收二氧化碳，合成尿素，作為額外的碳氮源來源。最後根據甘油碳流量的分析，解釋在甘油受限誘導時，提高甘油誘導量可以提高重組蛋白質產量。

Pichia pastoris is a kind of methylotrophic yeast, with a high potential in application of heterologous protein expression system. It has lots of advantages such as low culture cost, fast growth and high yield. Moreover, it has characteristics of post-translation modifications that are not available in prokaryotic expression systems. Thus, it is widely used to produce recombinant proteins. However, the AOX1 promoter (alcohol oxidase I promoter) is strictly regulated, and is induced to start and produce recombinant proteins only when toxic and flammable methanol is used as a carbon source. Therefore, the goal of this study is to develop a methanol-free induction method which is used to produce recombinant protein and improve the disadvantages. Based upon previous works, this study used strains expressing additional MXR1 are paired with the AOX1 promoter to express xylanase. The results confirmed that the MXR1-reprogrammed strains increased the production of recombinant protein under methanol induction. The methanol-free process was performanced by glycerol-starvation induction method, and successfully demonstrated the experiment of the fermentor cultures.The glycerol-starvation induction was attributed to the gene expression of transcription factor MXR1 and glycerol transporter I (glycerol transporter I, GT1), and the gene expression of carbamoyl phosphate synthetase I (carbamoyl phosphate synthetase I, CPA1). Under glycerol restriction, lack of carbon source results in the increase of CPA1 expression and recovering carbon dioxide as an additional source of carbon and nitrogen to synthesize urea. Finally, according to the analysis of glycerol carbon flow, it is explained that under the glycerol-starvation induction, increasing the amount of glycerol-starvation induction can increase the production of recombinant protein.