建立Saccharomyces cerevisiae 5-甲基四氫葉酸之評估方法

Abstract

葉酸代謝異常患者體內5,10-methylenetetrahydrofolate reductase (MTHFR)活性降低，故較易有葉酸缺乏的情形。過去文獻以化學法分析酵母菌中的葉酸型式與含量，發現Saccharomyces cerevisiae富含5-甲基四氫葉酸5-methyltetrahydrofolate (5-MTHF)，然而不同型式之葉酸在萃取與分析過程中可能會互相轉換，因此酵母菌是否為良好5-MTHF來源仍需進一步確認。本研究希望建立以葉酸需求菌株Lactobacillus rhamnosus分析S. cerevisiae總葉酸含量之平台，並分析葉酸與S-腺苷甲硫胺酸S-adenosylmethionine (SAM) / S-腺苷高半胱胺酸S-adenosylhomocysteine (SAH)比率之關聯性，藉此瞭解酵母菌中所含之葉酸是否具甲基轉移之生物活性。除此之外，藉由去除合成5-MTHF之基因MET13，比較基因去除前後總葉酸含量的差異，推測S. cerevisiae培養於YPD培養基產生的5-MTHF含量。實驗結果顯示野生型S. cerevisiae總葉酸含量顯著高於met13△突變株，且野生型菌株培養於YPD培養液在指數期產生之5-MTHF約為1098 µg / 100 g乾重、生長遲滯期產生之5-MTHF則約為715 µg / 100 g乾重，分別占總葉酸含量的1/4與1/3，故有潛力成為良好之5-MTHF補充品；此外S. cerevisiae在不同生長時期會產生不同之葉酸總量，指數期產生的總葉酸量顯著高於生長遲滯期；在葉酸與SAM / SAH比率關聯中，得知野生型S. cerevisiae葉酸產量雖較met13△突變株高，但SAM / SAH比率無顯著差異，然而不論是野生型或met13△突變株酵母菌，SAM / SAH比率在指數期皆顯著小於生長遲滯期。雖然實驗中無法明確獲得5-MTHF含量，但可透過met13△突變株葉酸測定的結果間接推測酵母菌中5-MTHF含量，未來也許可利用代謝體學等方法進一步瞭解分析酵母菌中5-MTHF含量測定方式。

5,10-Methylenetetrahydrofolate reductase (MTHFR) is responsible for the synthesis of 5-methyltetrahydrofolate (5-MTHF). The mutation of MTHFR reduces the activity of this enzyme and may cause folate deficiency. In the past, folate in yeast was measured by chemical analysis and the result showed that it was a good source of 5-MTHF. However, different types of folate may be converted during the extraction and analysis, so whether the yeast is a good source of 5-MTHF still needs further confirmation. In this study, the folate demanded strain Lactobacillus rhamnosus was used to analyze the total folate in Saccharomyces cerevisiae. This study also investigated yeast’s folate content in different growth periods and the correlation between folate and S-adenosylmethionine / S-adenosylhomocysteine ratio (SAM / SAH) for the biological methyl transformations. By removing the MET13 gene that synthesizes 5-MTHF, we could calculate the 5-MTHF content in wild type S. cerevisiae culturing in YPD medium via comparing the total folate content before and after gene removal. The results showed that the total folate content in wild-type S. cerevisiae was significantly higher than the met13△ mutant, and the 5-MTHF produced by wild-type S. cerevisiae in YPD medium at log phase and stationary phase were 1098 µg and 715 µg per 100 g dry weight, accounting for 1/4 and 1/3 of the total folate content respectively , so S. cerevisiae has the potential to become a good source of 5-MTHF supplement. In addition, S. cerevisiae had different folate contents in different growth periods, and the folate content at log phase was significantly higher than stationary phase. In the correlation between folate and SAM / SAH ratio, it was found that the folate production in wild type was significantly higher than met13△ mutant, but there was no significant difference in the SAM / SAH ratio. However, the ratio at log phase was significantly smaller than stationary phase in wild type and met13△ mutant. Although the content of 5-MTHF in yeast cannot be clearly obtained in the experiment, it can be indirectly inferred through the result of the folate content in met13△ mutant. In the future, it may be possible to use metabolomics or other methods to further understand the 5-MTHF content in yeast.