利用嗜甲醇酵母菌進行重組蛋白質表現之醱酵生產及分子調控之研究

Abstract

本研究利用醱酵生產的調控方式，提高嗜甲醇酵母菌Pichia pastoris及Pichia methanolica生產重組木聚醣酶之產量，並利用Saccharomyces cerevisiae的IRES建構嗜甲醇酵母菌雙順反子表現質體，於Hansenula polymorpha中成功地表現位於IRES下游的綠色螢光蛋白質。在醱酵生產的調控上，P. pastoris利用高密度細胞培養且在誘導前置換培養基，並維持誘導時甲醇濃度於0.5%可以得到最高的木聚醣酶活性，在甲醇誘導後10天木聚醣酶活性為5400 U/ml。而在P. methanolica中利用全合成培養基，並以1.8 ml/h/l固定流速連續添加甲醇的方式進行誘導，可以得到最高的木聚醣酶活性，在甲醇誘導後10天木聚醣酶活性為6200 U/ml。在P. methanolica誘導策略的研究上，固定流速連續添加甲醇的模式較依溶氧曲線逐段提高甲醇添加量及利用MC168維持甲醇濃度恆定的模式，在誘導時所需的的甲醇量最少而木聚醣酶活性最高，且菌體生長的數目最少。分析P. pastoris及P. methanolica表現的重組木聚醣酶可知兩者皆有不錯的熱穩定性及pH穩定性。利用SDS-PAGE及西方雜合法分析二者生產的重組木聚醣酶，可知其胞外上清液內的主要成分皆為木聚醣酶，以醣蛋白染色法可以確認二者所生產的重組木聚糖酶皆有糖基化修飾，然而此種糖基化修飾可能是較為複雜的O端糖基化修飾。總而言之，P. methanolica有較簡便的醱酵及誘導程序，並可以得到較高木聚醣酶活性，然而其胞外雜蛋白較多，而P. pastoris的生產的木聚醣酶活性稍低但胞外雜蛋白較少有利於後續的分析純化及應用。此外，利用P. methanolica之pulse-chase實驗可以觀察到在甲醇添加、鹼的添加及木聚糖酶活性上有一密切的關係。此外，為了能夠提高嗜甲醇酵母菌的應用性，本研究建構一個可以用於嗜甲醇酵母菌，以AOX1為啟動子並以木聚糖酶及綠色螢光蛋白質 (EGFP) 為報導基因，並在兩個基因之間加入一個可以替換不同來源IRES (internal ribosome entry site) 之MCS (multiple cloning site) 的雙順反子表現質體，測試來自Saccharomyces cerevisiae的p150、YAP1、TFIID、HAP4 之5’端未轉譯區的IRES序列。在H. polymorpha內發現來自YAP1、TFIID、HAP4之IRES皆具有活性，並成功地使IRES下游的EGFP表現。利用西方雜合法及螢光顯微鏡可明顯地觀察到EGFP訊號，然而來自p150之IRES在H. polymorpha內僅能使EGFP少量地表現，而在P. pastoris內並無法偵測得到EGFP的表現。

In this study, the xylanase gene from the rumen fungus Neocallimastix frontalis was expressed in Pichia pastoris and Pichia methanolica. In the study of methanol induction strategy of P. methanolica, the highest xylanase activity, fewest amounts of methanol feeding, and least cell growth was made by using continuous methanol feeding strategy. Using high cell density culture with medium replacement before induction and the maintenance of the methanol induction level at 0.5%, P. pastoris was able to produce about 5400 U/ml of xylanase after 10 days of induction. For P. methanolica, about 6200 U/ml of xylanase was reached after 10 days of induction using synthetic medium as first culture medium and then direct induction by continuous methanol feed at 1.8 ml/l/h. In general, the advantages of using P. methanolica to produce the xylanase were higher protein production, the lack of medium replacement, and an ease of scale up. However, due to the culture supernatant with fewer secreted non-target proteins, xylanase purification in P. pastoris would be easier than in the P. methanolica system. The recombinant xylanases from P. pastoris and P. methanolica were glycoproteins, which were in complex O-link glycosylation and had good thermostability and pH stability. In addition, methanol pulses-chase experiment suggested that these was a corelation among base feeding, methanol consumption and xylanase activity. In addition, in order to improve the application of methyltrophic yeast by dicistronic expression plasmid, a methyltrophic yeast dicistronic expression vector was constructed based on the 5’UTR internal ribosome entry site (IRES) of the Saccharomyces cerevisiae p150, YAP1, TFIID, HAP4. Expression vector containing the Neocallimastix frontallis xylanase gene and green fluorescent protein gene flanking the yeast 5’UTR IRES can produce recombinant xylanase and fluorescence in methyltrophic yeast Hansenula polymorpha under the control of alcohol oxidase1 (AOX1) promoter. After methanol induction for three days, the green fluorescence in H. polymorpha was observed under fluorescence microscope. From Western blot analysis, the expression of green fluorescent protein was confirmed. Unfortunately, Pichia pastoris containing this dicistronic plasmid with p150 5’UTR IRES can not produce green fluorescent protein. These results suggest that the S. cerevisiae p150, YAP1, TFIID, HAP4 IRES can be used in the development of dicistronic expression vectors for production of heterologous multiprotein complexes, or can be used for selection markers to facilitate the applications of H. polymorpha.