以非甲醇策略誘導漢遜氏酵母菌表現重組木聚醣酶

Abstract

嗜甲醇酵母菌Hansenula polymorpha是常用的異源蛋白質表現系統，可藉由強力且可調控的甲醇氧化酶啟動子 (methanol oxidase promoter, MOXp) 來啟動異源蛋白質表現。MOXp除了能使用甲醇進行誘導外，還能藉由甘油、葡萄糖、木糖為單一碳源之碳源受限 (carbon source starvation) 方式，使MOXp發生去抑制 (derepression) 作用進而表現下游基因。目前工業上嗜甲醇酵母菌大規模的醱酵應用，大多使用甲醇做為誘導物，但使用甲醇的缺點包括甲醇具有毒性及易燃性，對於工業化大量生產會增加風險及管控成本、濃度過高的甲醇對菌體造成毒性或造成蛋白質產物降解、生產醫藥性蛋白質可能有甲醇殘留的疑慮。因此，發展一個非甲醇誘導之誘導策略實屬必要。本研究以不同的溶氧模式調控甘油添加，使MOXp發生去抑制作用表現木聚醣酶。研究發現，以固定溶氧值之調控模式，溶氧值設定越低可得到較高的酵素活性，將溶氧值固定於80%、60%及40%，所生產的木聚醣酶最高活性分別為4368.30±85.65、6903.73±152.21和7512.47±147.13 U/ml (n=3)。並且在不同誘導模式中發現甘油的添加量、鹼的添加量及木聚醣酶活性增加量都呈現正相關，因此在醱酵過程中可做為重組蛋白質產量的良好指標。

The methylotrophic yeast Hansenula polymorpha is one of the most commonly used heterologous protein expression systems. Its methanol oxidase promoter (MOXp) is a strong and tightly regulated promoter, and can be used in heterologous protein expression. Except methanol, the MOXp can also be derepressed by single carbon source starvation such as glycerol, glucose or xylose for expressing downstream genes. Methanol is a commonly used inducer at large-scale fermentation of methylotrophic yeasts in industrial application. The disadvantages of using methanol include that methanol is toxic and flammable, the large-scale production of menthanol would increase the risk, and the cost for managing is high. In addition, high methanol concentration may hurt cells or cause target protein to be degrade. Since therapeutic proteins may face the risk of methanol remanet, it is necessary to develop a methanol-free induction strategy. In this study, using MOXp derepression to express xylanase through different pO2 control modes for regulating glycerol starvation. We found that lower pO2 control can get higher enzyme activity at fixed pO2 control.When pO2 control at 80%, 60% and 40%, the highest xylanase activity are 4368.30±85.65, 6903.73±152.21 and 7512.47±147.13 U/ml (n=3). And the addition of glycerol, base and xylanase activity correlated well with different pO2 control modes, suggesting that the glycerol and base addition can be a good indicator of recombinant protein production in fermentation course.