利用額外表達轉錄活化子Mit1搭配甘油受限誘導策略於畢赤酵母菌中生產葡聚寡醣氧化酶

Abstract

畢赤酵母菌(Pichia pastoris)為真菌異源蛋白質表現系統，可透過甲醇便宜、快速且大量的生產目標蛋白質，因此被廣泛利用於工業酵素的生產。AOX1啟動子(Alcohol oxidase 1, PAOX1)為畢赤酵母菌常被用於生產異源蛋白質的啟動子，然而PAOX1調控嚴謹，僅有甲醇誘導條件下可有效活化。甲醇具毒性與易燃性，限制其產物的應用，因此近年來非甲醇誘導逐漸受到重視。先前研究指出透過弱啟動子AOX2啟動子(Alcohol oxidase 2, PAOX2)額外表達轉錄活化子Mxr1，成功於甘油受限條件下提升PAOX1。Mit1為一種轉錄活化子，甲醇存在時會被大量誘導，並且直接結合於啟動子促使PAOX1活化。本研究使用GOOX(葡聚寡醣氧化酶)作為目標蛋白質，並利用PAOX2額外表達截短的Mit1(dMit1, RR1 RR2 deletion Mit1)，期望於低甘油濃度條件下額外表達dMit1，進而促進PAOX1活化，達到非甲醇誘條件下生產GOOX。首先由甲醇誘導實驗中，PAOX2可透過表達dMit1可促進PAOX1活化，搖瓶與發酵槽中產量分別提升1.67與1.38倍。接著於甘油的搖瓶誘導中，可觀察到GOOX產量提升為1.35倍，然而發酵槽甘油受限誘導的條件下，PAOX2無法生產足夠dMit1，導致觀察不到GOOX產量的提升。未來期望透過優化基因調控迴路，將PAOX2替換為強啟動子或具強去抑制活性的啟動子，以利於在微量甘油環境下表達dMit1，活化PAOX1，提供一個非甲醇誘導GOOX的方法。

Pichia pastoris is a fungal heterologous protein expression system. It can utilize methanol to express heterologous proteins efficiently and at low expense. It is widely used to produce industrial enzymes. The alcohol oxidase 1 promoter (PAOX1) is the most commonly used to produce heterologous protein in Pichia pastoris. PAOX1 is a strictly-regulated promoter, which is only activated under methanol induction. However, methanol is a toxic and flammable compound that limits its extensive application. Therefore, developing a methanol-free induction strategy is an emergent need. Previous study showed that using weak promoter PAOX2 to overexpress transcription activator Mxr1, successfully increased PAOX1 activation under glycerol-starvation conditions. Methanol-induced transcription factor 1 (Mit1) is a transcription activator that plays an important role in the activation of PAOX1. The existence of methanol highly increases Mit1 expression and subsequently activates PAOX1. In this study, we used PAOX2 to express mutant Mit1 (dMit1, RR1 RR2 deletion Mit1). To activate PAOX1 in low glycerol conditions. First, the methanol induction overexpressing dMit1 enhanced 1.67 and 1.38-fold of PAOX1 activity in the flask cultures and 2 L bioreactor fermentation. Second, the glycerol induction overexpressing dMit1 enhanced 1.35-fold PAOX1 activity in the flask cultures. However, under glycerol starvation induction in 2 L bioreactor fermentation condition, PAOX2 did not express enough dMit1 to activate PAOX1. This study provided a non-methanol induction method, and the optimizing the gene regulatory circuit still need further investigation in the future.