羥基癸酸（10-Hydroxydecanoic acid , HDA）於醫療、美容、食品、塑膠工業、化學溶劑等等各領域上具有廣泛的應用。然而羥基癸酸目前製備上仍對環境以及製程安全影響較大，因此本研究欲以代謝工程的方式生產化學品，不僅對環境友善且製程安全，相當具有發展潛力以及商業價值。
本研究中所使用的菌株為Candida viswanathii，HDA為該菌株之烷類代謝途徑的中間產物，本研究欲開發基因調控技術，以便於調控微生物基因之表現量，進而控制其代謝途徑，使最終產物集中在目標產物HDA。
CRISPR-Cas13d已被許多文獻指出會帶來嚴重的附帶效應，因此本研究欲開發對細胞影響甚小之基因調控技術，Csm與hfCas13d皆被研究指出能夠保有高效率的基因抑制，同時能夠減少對細胞的附帶效應，本研究欲將此兩種基因抑制工具應用於C.viswanathii中，透過螢光基因進行可行性的測試後，我們發現hfCas13d能抑制螢光基因表現量至56%，然而Csm並無明顯抑制效果。我們也觀察到Cas13d嚴重影響非標的基因的表現量，以及造成細胞生長遲緩的現象，反觀hfCas13d並無明顯之附帶效應。在相同發酵條件下使用hfCas13d基因抑制工具HDA產量能提高至23%，而Cas13d卻造成HDA產量下降，由此證實hfCas13d應用於生產HDA的代謝工程上，為更具有優勢之基因調控工具。
我們透過抑制PXA2以及ADH基因使HDA產量皆顯著提升，我們發現hfCas13d能夠抑制菌株內源基因約40～50%，並且最高可於搖瓶發酵中達到10.3 g/L的HDA產量。目前文獻中以生物轉化法生產HDA的最高產量僅有309 mg/L，而透過本研究的策略，我們成功將hfCas13d基因抑制工具應用於生產HDA當中，相較於HDA產量最高之文獻已大幅提升產量近30倍，為微生物轉化法生產HDA帶來突破性的發展。

10-Hydroxydecanoic acid (HDA) has extensive applications in medicine, cosmetics, food, plastics, and chemical solvents. However, current production methods for HDA have significant environmental and process safety impacts. Therefore, this study aims to produce HDA through metabolic engineering, which is environmentally friendly and safe in terms of production, showing considerable potential for development and commercial value. The strain used in this study is Candida viswanathii, with HDA being an intermediate product of its alkane metabolic pathway. This study aims to develop gene regulation techniques to control microbial gene expression, directing the metabolic pathway to concentrate the final product on HDA.

CRISPR-Cas13d has been reported in many studies to cause severe off-target effects. Therefore, this study aims to develop gene regulation techniques with minimal impact on cells. Both Csm and hfCas13d have been reported to retain high efficiency of gene inhibition while reducing off-target effects. This study intends to apply these gene inhibition tools to C. viswanathii. Through feasibility testing with fluorescent genes, we found that hfCas13d can inhibit fluorescent gene expression by up to 56%, while Csm showed no significant inhibitory effect. We also observed that Cas13d severely affects the expression of non-target genes and causes cell growth retardation. In contrast, hfCas13d exhibited no significant off-target effects. Under the same fermentation conditions, using the hfCas13d gene inhibition tool increased HDA production by 23%, whereas Cas13d caused a decrease in HDA production. This confirms that hfCas13d is a more advantageous gene regulation tool for metabolic engineering in the production of HDA.

By inhibiting the PXA2 and ADH genes, we significantly increased HDA production. We found that hfCas13d could inhibit endogenous genes in the strain by approximately 40-50%, achieving a maximum HDA yield of 10.3 g/L in shake flask fermentation. The highest yield of HDA reported in the literature using biotransformation methods is only 309 mg/L. Through the strategy developed in this study, we successfully applied the hfCas13d gene inhibition tool to HDA production, significantly increasing the yield nearly 30-fold compared to the highest reported literature yield, bringing a breakthrough in microbial biotransformation methods for HDA production.

第一章 文獻回顧...1
1-1羥基癸酸簡介...1
1-2 維斯假絲酵母菌（Candida viswanathii）...2
1-2-1菌株簡介...2
1-2-2 Candida viswanathii代謝途徑...3
1-3 基因調控工具的潛在缺點...5
1-3-1 CRISPR-Cas9...5
1-3-2 CRISPR interference...5
1-3-3 CRISPR-Cas13d...6
1-4 基因調控工具的開發...7
1-4-1 CRISPR-Csm...7
1-4-2 CRISPR-hfCas13d...8
1-4-3 gRNA的設計...9
1-5 研究動機...10
第二章 實驗材料與方法...15
2-1質體建構之方法...15
2-2 製作Candida viswanathii之勝任細胞...21
2-3 電穿孔質體...21
2-4 基因編輯Candida viswanathii...21
2-5 Colony PCR分析Candida viswanathii 菌落...22
2-6 定量即時逆轉錄聚合酶連鎖反應分析（qRT-PCR）...22
2-7 流式細胞儀...24
2-8 搖瓶發酵...24
2-9 產物分析...26
第三章 實驗結果與討論...32
3-1 驗證不同基因抑制工具在Candida viswanathii中的可行性...32
3-2 優化Culture Medium配方...33
3-3 優化Reaction Medium配方...34
3-4 選擇最佳基因抑制工具...35
3-5 評估基因抑制工具所帶來的附帶效應...37
3-6 選擇促進HDA生產之標的基因...39
3-7 利用hfCas13d基因抑制工具提升HDA產量...40
第四章 結論...50
第五章 未來展望...51
第六章 參考文獻...52

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