除了 C22:6n-3 (DHA) 及 C22:5n-6 (n-6DPA) 兩種主要的高度不飽和脂肪酸 (Highly unsaturated fatty acids, HUFAs) 外，橙黃壺菌 BL10 還含有包括 C22:5n-3 (n-3DPA)、C20:5n-3 (EPA) 及 C20:4n-6 (ArA) 在內的次要 HUFAs。這些次要的 HUFAs 在 BL10 進行低密度培養時的含量會顯著增加，然而其生合成路徑及生物意義均不清楚。本研究的目的，即在嘗試釐清這些問題。首先在比較不同培養條件的差異時，發現這些次要的 HUFAs 容易在低碳氮比及搖晃培養等容易促進細胞分裂，而較不利於油脂累積的培養條件中出現。再將 BL10 的基因體資料庫，經過基因搜尋及註解的結果，發現有 5 個疑似參與脂肪酸標準生合成路徑的基因： fads1、fads2、elovl1、elovl2和elovl3，能在其 putative proteins 中分別預測出 Δ-6 desaturase、ω-3 desaturase、Δ-9 elongase、C18-Δ-9 specific elongase 及 fatty acid elongase 的功能。進一步藉由前驅物添加實驗的做法探討 BL10 細胞內是否具有這些酵素的活性，由結果確認含有 Δ-6、Δ-5 兩種 desaturase 及 Δ-6、Δ-5 兩種 elongase 的活性。進一步的分析，探究 EPA 及 ArA 是否可能來自 DHA 及 n-6DPA 的 retroconversion，結果證明 EPA 及 ArA 兩種次要的 HUFAs 應該是 DHA 及 n-6DPA 這兩種主要 HUFAs 的 retroconversion 產物。

Aurantiochytrium sp. strain BL10 contains primary highly unsaturated fatty acids (HUFAs), C22:6n-3 (DHA) and C22:5n-6 (DPA), as well as secondary HUFAs C22:5n-3 (n-3 DPA), C20:5n-3 (EPA), and C20:4n-6 (ArA). Researchers have significant increased the content of secondary HUFAs under low-density culture conditions; however, the biosynthetic pathway and biological significance of these findings have yet to be elucidated. Our objective in this study was to clarify these issues. We found that the secondary HUFAs tend to accumulate in cells cultured a medium with a low carbon-nitrogen ratio under shaking. Annotation of the BL10 genome revealed five genes suspected to be involved in the standard synthesis pathway of fatty acids: fads1, fads2, elovl1, elovl2 and elovl3. The protein functional domains predicted by these genes are as follows: Δ-6 desaturase, ω-3 desaturase, Δ-9 elongase, C18-Δ-9 specific elongase and fatty acid elongase. Fatty acid precursor feeding experiments confirmed the activities of the two desaturases (Δ-6 and Δ-5) and two elongases (Δ-6 and Δ-5), which means that BL10 is able to use utilize exogenous fatty acids to synthesize C22:4n-6, C22:5n-3, and intermediates. We also determined that EPA and ArA may come from the retroconversion of DHA and n-6DPA, whereas n-3DPA is probably converted from EPA by Δ-5 elongase.

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