利用甘油受限誘導漢遜氏酵母菌表現重組蛋白質

Abstract

嗜甲醇酵母菌Hansenula polymorpha是目前常用的異源蛋白質表現系統之一，可藉由強力且可調控的甲醇氧化脢啟動子(methanol oxidase promoter, MOXp)來表達異源蛋白質。甲醇氧化脢啟動子可經由甲醇誘導，也可藉由甘油或木糖的去抑制作用(derepression)表達異源蛋白質；但會受到六碳糖、雙醣及乙醇的抑制。在大規模的醱酵應用上，利用甲醇誘導表現異源蛋白質仍有許多困難。例如：甲醇具有毒性及易燃性而需增設防護設備；高濃度的甲醇不利於菌體生長，也會造成蛋白質產物降解；代謝甲醇時，需額外增加通氣量。為了避免上述甲醇誘導量產異源蛋白質的困難，本研究欲建立一個非甲醇誘導但仍兼顧嗜甲醇酵母菌系統優勢的表現系統。 在本研究中，成功地利用溶氧控制甘油的添加，於H. polymorpha產生去抑制作用，表現選殖自Neocallimastix frontalis的木聚醣脢Xyn11B’與Ganoderma microsporum的免疫調節蛋白質GMI。溶氧調控模式所得木聚醣脢活性為2219.42±94.05 U/ml，與甲醇誘導模式的2236.10±8.43 U/ml相當，且醱酵時間可從192小時縮短為60小時；免疫調節蛋白質GMI濃度則達到187.56±10.98 mg/l。這個新的表達系統不僅保留了原始MOXp的特性，更成功地利用甘油為碳源進行誘導表現異源蛋白質。

The methylotrophic yeast Hansenula polymorpha is one of the most commonly used platform for heterologous protein expression due to its strong and expression regulatable methanol oxidase promoter (MOXp). The MOXp can be induced by methanol and partially derepressed by glycerol or xylose, as well as repressed in the presence of hexoses, disaccharides or ethanol. There exist several disadvatages such as extra facility required to use this toxic and flammable compound, slow growth, recombinant protein degradation and extensive aeration, etc. when using methanol-induction at large scale production. In order to solve the above problems, it is necessary to establish an alternative induction mode without using methanol. In this study, a new expression platform by using the derepression of H. polymorpha through the pO2 control was developed. A xylanase gene, xyn11B’, cloned from Neocallimastix frontalis and an immunomodulatory protein gene, gmi, cloned from Ganoderma microsporum were used to evaluate the derepression mode. The recombinant xylanase production reached 2219.42±94.05 U/ml after 60 h of induction by derepression in high cell density fermentation, compared to 2236.10±8.43 U/ml by methanol induction for 192 h. The expression level of GMI protein was 187.56±10.98 mg/l using the same approach. This improved expression platform prevented from the disadvantages of using methanol induction, while preserved the strong and regulatable nature of MOXp.