以轉錄因子Mxr1再程序化的策略加強嗜甲醇酵母菌 Pichia pastoris AOX1啟動子效率

Abstract

Pichia pastoris為嗜甲醇酵母菌的一種，是極具潛力的異源蛋白質表達系統，兼具微生物與真核系統的優勢。搭配甘油培養－甲醇誘導策略，可嚴謹調控P. pastoris的AOX1啟動子，並大量生產重組蛋白質。然而，調控過度嚴謹的AOX1啟動子，使P. pastoris只能侷限在特定的培養條件，必需以有毒、易燃的甲醇作為唯一碳源時，才可有效率誘導AOX1啟動子。本研究希望藉由轉錄因子再程序化，改變AOX1啟動子的調控，以提升P. pastoris的生產效率。透過建立轉錄活化子Mxr1 (Methanol expression regulator 1)的自我正回饋控制迴路(Positive auto-regulation circuit)，降低抑制性碳源對AOX1啟動子的干擾，並解決Mxr1稀釋效應所造成轉錄活性下降。以AOX1啟動子表現綠色螢光蛋白質基因，並以AOX2啟動子表現額外的Mxr1。在不同種類及濃度的碳源培養下，證實此策略可以提升AOX1啟動子的轉錄活性，並且不會造成細胞生長缺陷。在甘油高於特定濃度時，AOX1啟動子仍保留嚴謹的控制。但當甘油降至特定濃度後，Mxr1正回饋迴圈的啟動，逐步降低AOX1啟動子的嚴謹度，去除殘留抑制性碳源對AOX1啟動子的干擾，進而使AOX1啟動子能受甘油受限的誘導。然而，提升額外Mxr1之拷貝數，無法進一步加強AOX1啟動子的轉錄效率，顯示Mxr1與外源性AOX1啟動子的最佳比例仍需更深入目標的探討。最後，AOX1啟動子提升的轉錄活性，可能會受限於外泌效率，而影響胞外蛋白質的產量，但未來可以搭配其它策略解決此問題。總而言之，透過轉錄因子再程序化的策略，可以保有AOX1啟動子原有的優勢，加強甲醇誘導的轉錄活性，同時解決AOX1啟動子調控過於嚴謹的缺點，使P. pastoris更具應用性。

The methylotrophic yeast Pichia pastrois has been extensively applied in production of recombinant proteins because it combines the advantages of single cell in microbial and post-translational modification in eukaryotic systems. The AOX1 promoter (PAOX1) is the most common promoter used for heterologous protein expression in P. pastoris. A glycerol-methanol-shift induction strategy is applied to achieve high productivity. However, the tightly regulated PAOX1 also led P. pastoris expression to restrictive conditions. To improve the efficiency of protein production, we tried to reprogram the transcriptional regulation of PAOX1 in P. pastoris. The ectopic Mxr1 expressed by the mild AOX2 promoter (PAOX2) did not cause growth defect. The transcriptional efficiency of PAOX1 was enhanced since the limitation of Mxr1 titration effect was broken by extra Mxr1. PAOX1 became more flexible due to the positive feedback of Mxr1 and was regulated by glycerol. With the extra Mxr1 driven by PAOX2, PAOX1 showed better activity without than that with medium replacement. Moreover, glycerol starvation induced GFP production with reprogramming Mxr1 in P. pastoris. Increasing copy number of ectopic Mxr1 did not enhanced the efficeince of PAOX1. These results showed overexpression of Mxr1 by one copy of PAOX2 might be enough to achieve the maximum activity of PAOX1. Although the improvement of transcriptional efficiency might be limited by secretory ability, these problems could be sloved by combination with other strategies. In conclusion, transcriptional reprogramming of Mxr1 improved the efficiency of P. pastrois under methanol induction and potentially made P. pastrois become methanol-free induction system to eliminate the problems of methanol.