利用Pichia pastoris異源表現Saccharomyces cerevisiae之alcohol acetyltransferase I基因以生產isoamyl acetate

Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase I gene in Pichia pastoris for production of isoamyl acetate

陳姿伶(Tzu-ling Chen)

指導教授 ： 李綉鈴

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

Isoamyl acetate為發酵性酒精飲品之重要香味化合物，其由Saccharomyces cerevisiae之alcohol acetyltransferase (AATase)催化isoamyl alcohol與acetyl-CoA所生成。本研究乃將S. cerevisiae之ATF1 架構於一持續表現載體(pGAPZA)，轉殖進入P. pastoris GS115，以大量表現ATF1來生產天然芳香化合物isoamyl acetate，並探討表現ATF 1，生產isoamyl acetate之最適培養條件，冀能提高芳香化合物isoamyl acetate 之產量
吾人以不同的基質濃度isoamyl alchol探討其最適條件，發現當培養液額外添加20mM isoamyl alchol可獲得最高isoamyl acetate之含量，約為0.87 mM，而在碳源的試驗上則是以2 % glucose為最佳。Yeast extract之濃度試驗則發現，當yeast extract添加濃度為1~3 %時，其isoamyl acetate之含量則無顯著性差異。而在peptone之濃度試驗則發現，培養液之isoamyl acetate含量並不受到peptone濃度之影響。因此，根據本研究結果得到，當轉型酵母菌P. pastoris培養於含2% glucose、1 % yeast extrace與20 mM isoamyl alcohol時，可獲得最佳isoamyl acetate之產量，約為0.87 mM。

關鍵字

表現質體 ； 重組質體 ； 勝任細胞 ； 轉殖 ； 異源表現

並列摘要

Isoamyl acetate is an important flavor compound in wine and beverage.
It is formed from isoamyl alcohol and acetyl-CoA catalyzed by alcohol
acetyltransferase (AATase) in Saccharomyces cerevisiae. In this study, the
gene ATF1 coded for AATase from S. cerevisiae BCRC 21731 was cloned
and constructed into a constitutive expression vector pGAPZA. A
recombinant Pichia pastoris strain was obtained express high level natural
flavor to produce isoamyl acetate. The optimal culture condition to enhance
the production of isoamyl acetate by recombinant P. pastoris was also
investigated.
The results showed that recombinant strain GS115/GAPZA-ATF1
produced twice as much isoamyl acetate as GS115/GAPZA (control strain).
When glucose, other than glycerol or methanol, was used as carbon source,
recombinant strain produced the highest amount of isoamyl acetate. The
optimal medium composition for isoamyl acetate production by recombinant
strain was 2% glucose, 1% yeast extract and 20 mM isoamyl alcohol. The
highest production of isoamyl acetate, is 0.87 mM, was obtained when the
recombinant strain GS115/GAPZA-ATF1 was cultivated in the optimal
medium at 30℃ for 4 days.

並列關鍵字

alcohol acetyltransferase ； heterologous expression ； Pichia pastoris ； Saccharomyces cerevisiae

參考文獻

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