利用漢遜酵母MOX及FMD啟動子異源表現瘤胃真菌Neocallimastix frontalis之木聚醣酶

Abstract

嗜甲醇酵母菌係指一群可以甲醇為唯一碳源生長的酵母菌，包括Candida，Hansenula，Pichia及Torulopsis等菌屬，目前主要以H. polymorpha、P. pastoris和P. methanolica廣泛地應用於各種蛋白質的大量生產，如工業酵素及醫藥用蛋白質等。然而，由於菌種特性使然，P. pastoris以甲醇誘導前常須先置換培養基才能提高蛋白質產量，而P. methanolica雖然不須置換培養基，但所生產的胞外蛋白質因過於複雜而不利於純化回收。本研究旨在利用另一種嗜甲醇酵母菌，H. polymorpha之野生型菌株及P. pastoris之商品化載體經置換啟動子後，搭配抗生素耐受度之篩選策略以建立操作方便及篩選快速的H. poymorpha表現系統，並以厭氣真菌Neocallimastix frontalis之木聚醣酶為目標蛋白質，藉以評估此系統在表現異源蛋白質時，能否排除上述其他酵母菌系統所遭遇的問題。結果顯示，帶有木聚醣酶基因的轉形株不僅可直接添加甲醇誘導表現，其胞外蛋白質亦以木聚醣酶為主，其中以MOX為啟動子者，於搖瓶及醱酵槽培養之木聚醣酶最高活性分別為615.37±45.56及2236.10±8.43 U/ml；而以FMD為啟動子之表現活性則為170.32±16.51及447.61±18.31 U/ml。

Among yeasts, the genera of Candida, Hansenula, Pichia and Torulopsis are capable of growing on methanol as the sole carbon source, hence are named as methylotrophic yeasts. Many applications of H. polymorpha, P. pastoris and P. methanolica to produce recombinant proteins such as industrial enzymes and therapeutic proteins have been described. However, the replacement of medium to enhance expression level before methanol induction and the complicated extracellular proteins have limited the extensive applications of P. pastoris and P. methanolica, respectively. In this study, the wild type strain and the commercial vectors replaced by two promoters, accompanied by a selection strategy of antibiotic tolerance were conducted to develop an easily and rapidly manipulated H. polymorpha expression system. Moreover, the target protein, Neocallimastix frontalis xylanase, was exploited to evaluate that this system overcomes the problems described above or not. As the results, recombinant xylanase was released after methanol induction without replacement of medium and occupied the vast majority of extracellular proteins. The highest expression levels of xylanase in flask cultivation and fermentation were 615.37±45.56 and 2236.10±8.43 U/ml of MOXZα-xyn11B’ 1B2-17; 170.32±16.51 and 447.61±18.31 U/ml of FMDZα- xyn11B’ 1B15, respectively.