有些酵母菌在具有高濃度葡萄糖的條件下，即使氧氣充足，仍以發酵作用作為糖代謝主要的模式，這些酵母菌被稱為Crabtree positive酵母菌 (例如: Saccharomyces cerevisiae) ；而克魯維乳酸酵母菌 (Kluyveromyces lactis) 則是Crabtree negative的酵母菌，以呼吸作用作為主要的糖代謝模式。研究指出，Crabtree positive酵母菌的祖先最初主要依靠呼吸作用進行糖代謝作用模式，為適應環境而演化成主要以發酵作用的糖代謝模式。為了探究酵母菌如何轉換糖代謝模式，本實驗室已利用同源重組的方式，將來自S. cerevisiae的糖運輸蛋白質基因Hxt置入K. lactis (KB101) 中，藉此探討在增強攝取葡萄糖能力的狀況下對於K. lactis生長的影響。結果顯示在大量表達Hxt5或Hxt6基因的情況下能有效提升K. lactis在呼吸作用下的生長速率。因此本研究透過適應性演化 (adaptive evolution) 的方式篩選出發酵作用能力比較強的K. lactis 酵母菌，以了解增強葡萄糖攝取能力的狀況下對K. lactis以發酵作用作為生長條件的影響。本研究透過持續將K. lactis酵母菌菌株培養在無法進行呼吸作用的情況下，使菌株產生適應性演化，以觀察K. lactis酵母菌進行發酵作用的變化狀態。我已繼代培養共200次，並測定第0、50、100、150及200次繼代培養後的菌株以發酵作用作為主要糖代謝路徑的生長曲線，以觀察葡萄糖運輸能力對K. lactis酵母菌生長變化的影響。本研究在繼代培養200次後的適應性演化實驗中，也得到18個生長快速的 KB101 (Hxt5) 單一菌落； 11個生長快速的 KB101 (Hxt6) 單一菌落。我從KB101 (Hxt5) 及KB101 (Hxt6) 品系中各挑選5個菌株進行生長曲線測定，實驗結果顯示適應性演化中出現的生長快速菌株其發酵作用下的生長速率與未經適應性演化的原始菌株相比皆顯著提升。然而某些適應性演化後的整體族群其生長速率與未經適應性演化的原始菌株相比皆呈現下降現象。後續觀察發現隨著適應性演化次數的增加，K. lactis酵母菌細胞會逐漸出現絮凝現象，以測量OD600的方式來呈現菌株生長速率就會產生誤判情況，推測不同繼代培養次數所造成的細胞聚集程度並不相同，因此造成菌液中的懸浮菌數不易被精準估計出來。在未來實驗設計中，可以設計其他方式來探討生長速率的改變，並可以透過RNA定序的方式來了解這些經適應性演化後發酵作用能力變強的K. lactis酵母菌有哪些基因的表現量受到影響。

Under high glucose condition, some yeasts, such as Saccharomyces cerevisiae, utilize fermentation as their primary metabolic process even if there is sufficient oxygen. These yeasts are known as Crabtree positive yeasts. In contrast, Kluyveromyces lactis is a Crabtree negative yeast, primarily relying on respiration process for sugar metabolism. Previous researches suggest that the ancestors of Crabtree positive yeasts might initially depended on respiration for sugar metabolism and later evolved to adopt fermentation under high level of glucose conditions even if there is sufficient oxygen. Our laboratory previously introduced several hexose transporter genes (Hxts) of S. cerevisiae into K. lactis (KB101) to explore the impact of enhanced glucose uptake capability on the growth of K. lactis. Our results showed that overexpressing Hxt5 or Hxt6 genes significantly improved the growth rate of K. lactis. I now aim to study the fermentation capabilities of these K. lactis strains. In this study, K. lactis was grown on media with antimycin A to inhibit the respiration capability and selected for higher fermentation capability of K. lactis for a long period of time. In this study I found 18 fast-growing strains of KB101 (Hxt5), and 11 fast-growing strains of KB101 (Hxt6). I tested the growth rate of 5 fast growth KB101 (Hxt5) strains and 5 fast growth KB101 (Hxt6) strains and found that these strains do grow fast than the parental strains.

潘冠吾，(2022)。大量表達六碳糖轉運蛋白質對於克魯維乳酸酵母菌生長之研究。國立成功大學生命科學研究所碩士學位論文。取自https://hdl.handle.net/11296/hpfpt4
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