探討 Saccharomyces cerevisiae kre6Δ 突變株及Dekkera bruxellensis 對羅漢果皂苷轉化之機制

Study on mogrosides bioconversion mechanism of Saccharomyces cerevisiae kre6Δ mutants and Dekkera bruxellensis

王如邦(Reuben Wang)

指導教授 ： 羅翊禎

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2016年)

https://doi.org/10.6342/NTU201601418

摘要

酵母菌(Saccharomyces cerevisiae)可透過生物轉化將羅漢果皂苷主要形式的五醣皂苷(mogroside V，MG V)轉換成兩種帶有四醣的siamenoside I (SI)、mogroside IV (MG IV)及一種帶有三醣的mogroside III E (MG III E)皂苷。過去研究顯示酵母菌Exg1 蛋白(ScExg1)是酵母菌發酵羅漢果皂苷之必要酵素，而當缺乏KRE6 基因(kre6 Δ 突變株)時可觀察到MG V 加速轉化為MGIII E 的現象。但其機制仍然未知，本研究將針對kre6 Δ 突變株快速轉化羅漢果皂苷機制進行探討。結果顯示ScExg1 仍是kre6Δ 細胞中負責轉化皂苷的酵素，且基於ScExg1 基因表現量不變下，kre6Δ 細胞有更多ScExg1 溢散於細胞外，是造成較快轉化皂苷醣基的主因。而分配更多ScExg1 到細胞外之特性，推測可能與kre6Δ 細胞之細胞壁異常有關。進一步透過選擇其他細胞壁突變株進行各別菌株之多種細胞壁結構分析實驗與皂苷發酵分析實驗，我們歸納出只有無法將細胞壁表面之甘露糖蛋白固定在細胞壁上之特定突變株，會造成甘露糖蛋白溢散在培養基中，並伴隨較快羅漢果皂苷轉化特性，促進MG III E 產生。另一方面，甜味及甜度更好的羅漢果皂苷S I，為目前天然甜味劑的生產目標物。然而過去研究受限於複雜的羅漢果皂苷醣基支鏈結構，無法找到有效增加萃取物中S I 比例的方法。透過菌種篩選，我們最終找出酵母菌Dekkera bruxellensis 所分泌的外泌酵素複合體中之glucan-β-glucosidase precursor (DbExg1)酵素，具專一性水解羅漢果皂苷C 3 上之β-1, 6 鍵結的葡萄糖醣基，將主要形式的MG V 完全轉換為S I。雖然ScExg1 與DbExg1 有高達55%胺基酸序列相似度，但卻分別產生MG III E 及SI。透過分析兩酵素演化史可發現兩者位於演化樹上之不同簇。依照演化樹分類對其他種類酵母菌進行羅漢果皂苷發酵測試，發現演化與皂苷產物高度相關。最後將DbExg1 基因崁入生長快速的S. cerevisiae 細胞之染色體中，取代原有ScExg1 基因，穩定且有效提升S I 的產生效率。

關鍵字

Saccharomyces cerevisiae ； 生物轉化 ； Mogroside III E ； Siamenoside I ； EXG1 ； KRE6 ； Dekkera bruxellensis

並列摘要

Yeast (Saccharomyces cerevisiae) can bioconvert the main mogroside, mogroside V (MG V) into siamenoside I (SI), mogroside IV (MG IV), and mogroside III E (MG III E) during fermentation. Previous study had shown that yeast Exg1 (ScExg1) was an indespensible protein for its mogroside bioconversion; and the deletion of KRE6 (kre6 Δ mutants) may results in an accelerative mogroside conversion phenotype, but the mechanism was still unknown. The aim of this study was to reveal the facilitative mogroside bioconversion of kre6 Δ mutants. The results showed that ScExg1 was still the enzyme responsible for the bioconversion of mogrosides in kre6Δ cells; and kre6Δ cells could released more of its ScExg1 into the media based on the same ScEXG1 expression levels relative to wild-type cells which was mian reason for its accelerative mogrosides bioconversion property. We proposed that more of ScExg1 allocation to extracellular region in kre6Δ cells was caused by its cell wall mutations. Further correlation analysis between cell wall structure mutations and mogrosides conversion abilities in different cell wall mutants, revealed that only specific cell-wall structure mutation which showed unfixed mannoprotein layer, with the unfixing mannoproteins diffused into the medium, was accompanied by higher mogroside conversion rates. In addtion, the better tasting and sweetest mogrosides, S I, is the current natural sweetener production target compound. However, due to the complexity of the glycosylation structure on mogrosides compounds, no effective methods have been developed which effectivtly increase in the proportion of S I in the mogroside extracts. Through intensive screening processes, we found that the yeast glucan-β-glucosidase precursor (DbExg1) enzyme from Dekkera bruxellensis, could specifically hydrolyze the glucose moiety with β-1, 6 linkages at C3 position of mogrol. This hydrolytic specificity led to the complete conversion of MG V to SI. Although ScExg1 and DbExg1 have 55% of amino acid sequence similarity, they generate MG III E and S I as end product respectively. Through analyzing the enzyme’s evolution history by constructing the evolutionary tree, we found these two enzymes were in different clusters. We also found that the mogrosides conversion specificity of cells were highly relevant to their classification results in the evolutionary tree. Finally, we integrated DbEXG1 to the chromosome of fast growing yeast, S. cerevisiae, to replace its original ScEXG1. This helped to effectively increase the bioconversion efficiency of mogrosides and produce more S I base on same fermentation time.

並列關鍵字

Saccharomyces cerevisiae ； bioconversion ； mogroside III E ； siamenoside I ； KRE6 ； EXG1 ； Dekkera bruxellensis

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