利用添加異戊二烯鏈之前驅物與促進內酯化反應提升牛樟芝菌絲體 4-AAQB 之產率

Abstract

4-acetylantroquinonol B (4-AAQB) 及 Antroquinonol (AQ) 為牛樟芝菌絲體中最具代表性的兩個泛醌類成分，過去之研究顯示 4-AAQB 比AQ具有較強的抗肝癌效果，故本研究之目的為提升 4-AAQB 之產率。從生合成途徑來看 4-AAQB 及 AQ 生合成的分界點為六碳環的 farnesylation，4-AAQB 側會接上帶有 γ-lactone 的 FPPB，AQ 側則是接上 FPP，而本研究提出之假說為 FPP 經由 Mevalonate pathway 形成後，再經由 lactonization 形成 FPPB，因此，若能提高 FPP 轉化為 FPPB 的能力則能提升 4-AAQB 產率，並同時降低 AQ 產率。相關研究顯示 γ-lactone 的形成需要在酸性環境下進行。因此本研究嘗試以調酸來提高 4-AAQB 產率，並且透過添加 Farnesol 作為前驅物與調酸共同作用而達到加乘效果。結果顯示，在發酵第 14 天將發酵液調酸至 pH 2.75 可以顯著提升 4-AAQB 產率並降低 AQ 產率，且對於菌絲體的生長沒有負面影響。以 LC MS/MS 分析生合成途徑中間產物，結果顯示，調酸可以促進 5-DMQ0 形成 CoQ3B 以及 5-DMQ0 形成 CoQ3 的步驟，而從 CoQ3B 形成 4-AAQB以及 CoQ3 形成 AQ 的步驟則沒有受到影響。此外，調酸對於 4-AAQB 側以及 AQ 側六碳環的 farnesylation 都有提升的現象，藉此推斷調酸會增加上游 FPPB 的形成。另外，添加 0.1% farnesol 會同時提升 4-AAQB 側以及 AQ 側 farnesylated 的中間產物，但對於 AQ 側六碳環修飾步驟卻有降低的影響，特別是 CoQ3 形成 AQ大幅降低了95%，而添加 farnesol 對 4-AAQB 側影響較小，只有 AQB 形成 4-AAQB 的步驟略為降低。因此，添加farnesol最終導致 AQ 之產率大幅降低，而 4-AAQB 之產率卻沒有受到影響。由以上結果可以推論，FPPB 極有可能是由 FPP 經 lactonization 而來，因為添加 farnesol 後 4-AAQB 側以及 AQ 側 之 farnesylated 中間產物都大量提升了，而調酸確實可以促進 FPPB 的形成，後續若適當的結合調酸以及添加 farnesol，則有機會大幅提升 4-AAQB 之產率。

4-acetylantroquinonol B (4-AAQB) and Antroquinonol (AQ) are two of the most iconic ubiquinones from the mycelium of Antrodia cinnamomea (AC). 4-AAQB has been proven to be more effective in inhibiting the growth of liver cancer cells than AQ. Therefore, the objective of this study was to increase the yield of 4-AAQB during cultiva-tion of AC. The dividing step in the biosynthetic pathways of 4-AAQB and AQ is farne-sylation of benzoquinone rings. The benzoquinone rings are either farnesylated with far-nesylpyrophosphate (FPP) or FPPB, the suffix B represents γ-lactone modification at the end of FPP. The hypothesis of this study was that FPPB are formed by lactonization of FPP. Hence, we supposed that if the lactonization process could be accelerated, then the yield of 4-AAQB would be increased and the yield of AQ decreased. Since acidic condi-tion favors the formation of γ-lactone, the effect of pH adjustment of the culture medium on the yields of 4-AAQB and AQ were investigated, and the effect of addition of farnesol as a precursor to FPP and FPPB was also studied. We expected that combination of these two treatment would further increase the yield of 4-AAQB. UPLC quantitative analysis has shown that adjusting the pH of the broth to 2.75 at the 14th day during fermentation could significantly increase the yield of 4-AAQB and decrease the yield of AQ. Further analyses of the biosynthetic intermediates by LC MS/MS revealed that farnesylation and O-methylation of the intermediates for the biosynthesis of both 4-AAQB and AQ were promoted by pH adjustment, but ketone group reduction and acetylation of benzoquinone rings were not affected. This phenomenon could mean that the reaction of FPP lactonized to FPPB was promoted. LC MS/MS analyses also showed that addition of 0.1% farnesol could increase farnesylation steps for the synthesis of both 4-AAQB and AQ, but it strongly inhibited the benzoquinone rings modification on AQ side of biosynthetic path-way, especially the conversion of CoQ3 to AQ. On the other hand, addition of farnesol did not affect too much on 4-AAQB side of biosynthetic pathway. Consequently, addition of farnesol during fermentation significantly reduced the yield of AQ but did not affect the yield of 4-AAQB. The results of this study suggested that pH adjustment could promote lactonization of FPP to formed FPPB. It seems promising to find a proper way to adjust pH and add farnesol at the same time to further increase the yield of 4-AAQB.