在 Komagataella phaffii 中以同源重組策略提升 CRISPR/Cas9 基因編輯效率

Abstract

嗜甲醇酵母菌 Komagatealla phaffii (Pichia pastoris) 為目前被廣泛使用之重組蛋白質表現系統，可以甲醇誘導之強力啟動子 AOX1 大量表現重組蛋白質，且具有與哺乳動物較為相近之轉譯後修飾及可以釀酒酵母之外泌訊號胜肽α因子將蛋白質分泌至胞外之特性，常被使用於科學研究及工業發酵領域。 近年來隨著合成生物學之發展， 同時針對 Komagatealla phaffii 蛋白質表現系統內不同因子進行調控以及建構多個基因迴路之研究逐漸盛行，希望能以各式策略強化 K. phaffii 系統之各種優勢，使其能具備更高之應具價值。然而目前 K. phaffii 系統中已發表之合成生物學常見工具，如基因編輯系統、基因篩選標記回收系統、胞內 DNA 組裝工具及基因調控系統皆較為缺乏，妨礙此類研究的大幅進展。 本研究首先以前人於 K. phaffii 內建構之以 AOX1 啟動子表達 Cas9 及以 U6 啟動子表達 sgRNA 之 CRISPR/Cas9 工具將 K. phaffii 內生性基因 ADE2 及外源性基因 ZeoR 進行剔除，證明以該系統進行基因編輯之可行性。再以鼠李糖 (Rhamnose) 誘導之 LRA3 啟動子取代 AOX1 啟動子表達 hspCas9，避免影響 AOX1 啟動子之重組蛋白質表達量，並配合在質體上加入目標基因之同源重組模板及改變誘導表現 hspCas9 之流程，以提升 CRISPR/Cas9 基因編輯工具之效率，進一步改善本實驗室建構之 U6 CRISPR/Cas9 基因編輯工具之應用性。

The methylotrophic yeast Komagataella phaffii (Syn. Pichia pastoris) is a recombinant protein expression system who has a high expression level methanol inducible promoter AOX1, an post-translational modification similar to human cell in eukaryotic systems and ability to secret recombinant protein by Saccharomyces cerevisiae α-factor. These advantages make it widely used in industrial and research field. Along with synthetic biology become popular in recent years, there is more and more research aimed to regulated different factor and establish more complicated gene circuit. Try to improving K. phaffii’s capability and value. However, because the lack of some basic synthetic biology tools, such as genome editing tools, marker recycle tools, in vivo DNA assemble tools and gene regulation system, this type of research is still obstructed. This thesis wants to continue the previous research which establishes a CRISPR/Cas9 genome editing system in K. phaffii used AOX1 promoter express hspCas9 and endogenous U6 promoter express sgRNA. First of all, we choose K. phaffii 's auxotrophy marker gene ADE2 and drug resistance mafrker gene ZeoR as a target gene to knock out by the CRISPR/Cas9 system. Then we replace the AOX1 promoter by a rhamnose-inducible promoter LRA3 to prevent this CRISPR/Cas9 system impact the recombinant protein's yield. At last, we combine two strategies, an episomal plasmid contain homologous DNA Donor and improve the induction process, together to increase the CRISPR/Cas9 genome editing efficiency. Make the K. phaffii U6 CRISPR/Cas9 genome editing tool more applicability.