以E2-Crimson作為模式蛋白質探討CRISPRa Mxr1與CRISPRi Nrg1在Pichia pastoris調控AOX1啟動子效率

Abstract

Pichia pastoris (Komagataella pastoris) 為一種嗜甲醇酵母菌，因為生長迅速、低成本及具備原核細胞無法進行的轉譯後修飾，為廣泛應用異源基因表現系統的。P. pastoris表現系統最常使用之啟動子為AOX1啟動子 (alcohol oxidase I promoter)，該啟動子抑制子如Nrg1與活化子如Mxr1等轉錄因子調控，僅在使用甲醇為碳源時大量表現重組蛋白質。本研究希望藉由前人建構之基因調降系統CRISPRi (Clustered regularly interspaced palindromic repeats- based interference)針對Nrg1進行抑制與基因活化系統CRISPR activation來提升Mxr1表現，以增加AOX1啟動子的表現效率，並希望進一步以CRISPRi/a同時抑制Nrg1並提升Mxr1探討是否可以獲得更高啟動子表現效率，並以E2-Crimson四聚體紅色螢光蛋白質作為模式蛋白質，該蛋白質具低生物毒性、穩定及方便觀察。結果顯示，在單獨以CRISPRi抑制Nrg1或以CRISPRa提升Mxr1實驗組別可以增加更多的螢光蛋白質產量，經生菌數對數標準化處理後在搖瓶比控制組表現量分別提高0.4倍與0.28倍，且在醱酵實驗比控制組表現量分別提高0.43與1.1倍；當同時調控Nrg1及Mxr1時，不論CRISPRa與CRISPRi建構先後順序，經生菌數對數標準化處理後螢光蛋白質表現量，在搖瓶中比單獨抑制Nrg1提高0.57倍，也比單獨提升Mxr1提升0.5倍。在醱酵槽中測試中，總螢光量在誘導第一天與控制組相比最高達到2.39倍總螢光量，經生菌數或OD600標準化處理後螢光數值仍有相同趨勢，顯示抑制Nrg1與提升Mxr1具有協同作用效果。此外， Mxr1與Nrg1之mRNA表現量證實CRISPRi/a可有效調控轉錄因子表現量。AOX活性在同時調控Nrg1與Mxr1比控制組增加0.61倍活性。目前實驗結果顯示可以CRISPRi/a同時調控Nrg1及Mxr1，未來可以發展應用在不同目標蛋白質上，以提高目標蛋白質產量。

Pichia pastoris (Komagataella sp.), a methanolophilic yeast, is widely used in academic and industrial production because of its rapid growth, low cost, and post-translational modifications. The most promoter used in the P. pastoris expression system is the AOX1 promoter (alcohol oxidase I promoter), which is restrictively regulated by the transcription factors like repressors Nrg1 and activators Mxr1. Recombinant proteins are abundantly expressed when the carbon source is methanol. In this study, we use the Clustered regularly interspaced palindromic repeats-based interference (CRISPRi) and CRISPR-based activation (CRISPRa) that was constructed by previous researcher for regulating the activator Mxr1 and repressor Nrg1 expression. Hence, the PAOX1 efficiency was tested by using only CRISPRi/a or CRISPRi/a together, and evaluated by the expression of red tetrameric fluorescent protein (E2-Crimson) which has lower toxicity and stability. Our results demonstrate that the E2-Crimson expression enhanced by Nrg1 Knockdown or activation of Mxr1. The Normalized fluorescence increase 0.4 and 0.28 times in flask, and significant increases 0.43 and 1.1 times in bioreactor. Furthermore, the E2-Crimson have significant increment during Nrg1 Knockdown and activation of Mxr1 in flask or bioreactor induction. We also show that Mxr1 and Nrg1 mRNA was successfully regulated by CRISPRi/a or CRISPRi/a together, and the AOX1 activity results indicate that two CRISPRs together can increase significantly. The current results illustrate that CRISPRi/a can simultaneously regulate Nrg1 and Mxr1, and it can be developed to others target proteins in future for improving the yield.