橙黃壺菌L-BL10 (Aurantiochytrium sp. strain L-BL10) 富含22碳6烯酸 (C22:6ω3, DHA) 和22碳5烯酸 (C22:5ω6, DPA)，然18碳及20碳系列的不飽和脂肪酸含量極低。由過去本實驗室的研究，在其培養基中添加脂肪酸合成酶 (fatty acid synthase, FAS) 抑制劑後，DHA及DPA的產量不減反增的結果推測，L-BL10細胞內存有一個獨立於標準路徑以外的多元不飽和脂肪酸生合成路徑。因此本研究目的即想探討在此條特殊的生合成路徑中多元不飽和脂肪酸可能透過何種酵素進行催化。首先，由實驗室過去已解序出的L-BL10基因體序列搭配NCBI資料庫比對，找出和已知PUFAs (polyunsaturated fatty acids) 生合成基因近似的基因片段並將其完整解序。接著利用SMART (Simple Modular Architecture Research Tool) 軟體預測其功能結構域 (functional domain) 的種類，並由其排列方式與功能結構域的親緣性將此酵素進行分類，另一方面也利用即時聚合酶鏈鎖反應 (real-time PCR)，研究各個培養時間點其基因表現量的變化。
利用上述方法，本研究在L-BL10基因體中找到8段與Schizochytrium sp. ATCC 20888 合成DHA及DPA 的聚酮合成酶 (polyketide synthase, PKS) 類似的基因序列。ATCC 20888在過去證實其聚酮合成酶分別由三段基因所組成，分別命名為pksA、pksB及pksC。由本研究定序結果，分別獲得3個近似於ATCC 20888的pksA、pksB及pksC的完整基因序列，其長度分別為10,068、6,117及4,386 個鹼基對，且利用SMART軟體分析後找到合成脂肪酸所必須的功能性區域，如: ketoacyl synthase、malonyl-CoA:ACP acyl transferase、acyl carrier protein、ketoacyl-ACP reductase、enoyl reductase、chain length factor、acyl transferase和dehydrtase的功能性區域，顯示L-BL10的聚酮合成酶基因應具有合成長鏈不飽和脂肪酸的能力，並由其功能結構域排列方式與KS結構域親緣性分析，皆可將L-BL10的聚酮合成酶歸類在Type I iterative PKS。此外在基因表現分析結果中，發現聚酮合成酶基因在L-BL10的對數成長末期 (接種後20小時) 後才會開始大量表現，而此時L-BL10耗盡培養基內的氮源並開始消耗碳源。由此推測，L-BL10的聚酮合成酶基因表現可能受到營養源的調控，當碳氮源同時存在時，聚酮合成酶的基因並不會啟動，然一旦氮源消耗殆盡僅剩碳源時，其基因就會被誘導開始大量表現，進行DHA和DPA的累積。

Aurantiochytrium sp. strain L-BL10 is rich in C22 polyunsaturated fatty acids (PUFAs), DHA (C22:6n-3), and DPA (C22:5n-6); however, this species contains very few PUFAs with 18 and 20 carbons. Previous studies in our laboratory demonstrated that adding fatty acid synthase (FAS) inhibitor to a L-BL10 culture failed to decrease the percentages of DHA and DPA. These earlier results revealed that, in addition to the standard pathway, a secondary biosynthesis pathway is involved in the production of these two PUFAs in L-BL10. The purpose of our current research was to identify enzymes capable of catalyzing the synthesis of PUFAs along this specific biosynthetic pathway. First, several genes suspected of being involved in PUFA production were synthesized from the L-BL10 genome sequence and NCBI database. We then predicted the functional domains using SMART (Simple Modular Architecture Research Tool) software and investigated the enzyme classification according to functional domain arrangement and phylogenetic analysis. In addition, we examined gene expression over various incubation times using real-time PCR.
These efforts revealed that 8 polyketide synthase (PKS)-like genes may be involved in the production of DHA and DPA in Schizochytrium sp. ATCC 20888. Previous researchers confirmed that the polyketide synthase of ATCC 20888 comprises three genes, pksA, pksB, and pksC. The results of this current study further demonstrated that L-BL10 has three complete polyketide synthase genes, which are similar to Schizochytrium sp. ATCC 20888. The lengths of these genes are 10068, 6117, and 4386 base pairs, respectively. We also identified the functional domains capable of synthesizing PUFAs, such as ketoacyl synthase, malonyl-CoA:ACP acyl transferase, acyl carrier protein, ketoacyl-ACP reductase, enoyl reductase, chain length factor, acyl transferase, and dehydratase functional domain. These results indicate that L-BL10 should have the ability to synthesize PUFAs. Moreover, the PKS of L-BL10 was shown to belong to Type I iterative PKS according to its arrangement of functional domains and the results of phylogenetic analysis related to KS domains. Conversely, we found that PKS gene expression reached the culminating point when L-BL10 was in the last stage of log phase (after culturing for 20 hours). At this point, the nitrogen source on the culture plate was depleted and L-BL10 began using the carbon source. Thus, we surmise that the PKS gene may be regulated by its nutrient source, such that the co-existence of a carbon source and nitrogen source on the culture plate prevents induction of the PKS gene. Nonetheless, these conditions could promote the accumulation of DHA and DPA when induced by only a carbon source on the culture plate.

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