以CRISPRi及CRISPRa策略加強Komagataella phaffii (Pichia pastoris) AOX1啟動子效率

Abstract

Pichia pastoris是一種嗜甲醇酵母菌，因其生長快速、低成本、可大量表現蛋白質，且具有在原核細胞無法進行的轉譯後修飾等特性，因此常用來大量表達異源蛋白質。而其AOX1 啟動子 (alcohol oxidase I promoter) 因具有高度調控嚴謹性，且在被甲醇誘導後可大量產生目標蛋白質，因此在P. pastoris中為最常用來表現蛋白質之啟動子。本研究希望藉由調控AOX1啟動子之轉錄因子表現量，來提昇AOX1啟動子表現效率，進而增加目標蛋白質表現量。本研究將以活化子Mxr1及抑制子Nrg1做為切入點，利用基因調降系統CRISPRi (clustered regularly interspaced palindromic repeats -based interference) 抑制其抑制子Nrg1，並用基因活化系統CRISPRa (CRISPR-based activation) 提升其活化子Mxr1，並以AOX1 啟動子表現綠色螢光蛋白質 (mEGFP) 作為評估方式，希望藉由降低抑制子Nrg1或提升活化子Mxr1表現量，進而提升AOX1 啟動子綠色螢光蛋白質表達效率。結果顯示分別以CRISPRi抑制Nrg1抑制子及以CRISPRa提升Mxr1活化子確實皆可提升AOX1 啟動子之螢光蛋白質表現量，其中Nrg1 mRNA降低為控制組之0.5倍；而Mxr1 mRNA可提升為控制組之4~6倍，也驗證CRISPRi與CRISPRa基因調控工具於P. pastoris之可行性。此外，本研究另進行同時表達CRISPRi與額外表現Mxr1之協同實驗，且證實可使AOX1啟動子效率較單一方法之下率更高，螢光表現更大量。

Pichia pastoris (reclassified as Komagataella phaffii) is commonly used as expression system for heterologous proteins. The strictly-regulated and methanol inducible AOX1 promoter (PAOX1) is the most extensively used for proteins expression in P. pastoris. Several transcriptional factors are involved in the regulation of PAOX1 including the transcriptional activator, MXR1, and the repressor, NRG1. In this study, the PAOX1 efficiency was enhanced by clustered regularly interspaced palindromic repeats -based interference (CRISPRi) to downregulate Nrg1 and CRISPR-based activation (CRISPRa) to upregulate Mxr1 expression. Evaluated by the expression a monomeric enhanced green fluorescent protein (mEGFP) gene, the Nrg1 knockdown by CRISPRi successfully blocked the RNA polymerase initiation or elongation while transcription. In addition, the activation of Mxr1 by CRISPRa recruited more transcriptional activators to enhance gene expression. Our results show that the mEGFP expression enhanced by Nrg1 knockdown reached the maximum level 1 day earlier than that of the non-Nrg1 repressed strains. Similar results were also observed in the Mxr1 activation strains by CRISPRa to extend guide RNAs to include effector protein recruitment sites, and to recruit MCP-VP64 fusion protein. Consequently, VP64 recruited more transcription activators to help transcription of Mxr1. This CRISPRi and CRISPRa gene regulation platform provides a simple approach for regulate gene expression on in P. pastoris.