牛樟芝液態發酵之色素表現與 AQ 及 4-AAQB 生合成之關係

Abstract

牛樟芝為臺灣原生的藥用真菌。多種具有生物活性的次級代謝物蘊藏於牛樟芝菌絲體，其中屬於泛醌類似物的 antroquinonol (AQ) 與 4-acetylantroquinonol B (4-AAQB) 具有優異的抗肝癌功效而受到矚目。過去的研究發現，牛樟芝進行液態深層發酵時，AQ 及 4-AAQB 的產量可能與菌絲體的色素表現有正相關性。本研究嘗試在牛樟芝液態發酵培養基中添加 Cu2+、Zn2+ 及 4-nitrobenzoate (4-NBA)，藉由添加物的影響，探討菌絲體的色素表現與 AQ 及 4-AAQB 生合成途徑的關係。從色素表現上來看，添加 10 ppm Cu2+ 改變菌絲體的色素組成，可見光吸收圖譜產生紅移；添加 1 ppm Zn2+ 及 100 ppm 4-NBA 降低菌絲體色素的生合成，且 4-NBA 對色素表現的抑制具有劑量效應。添加 10 ppm Cu2+、1 ppm Zn2+ 及 100 ppm 4-NBA 皆能增進 4-AAQB 的產率；然而 1 ppm Zn2+ 與 100 ppm 4-NBA 卻抑制 AQ 的生合成。透過 UHPLC-MS/MS 分析添加物對 AQ 及 4-AAQB 生合成途徑的影響，結果顯示添加 10 ppm Cu2+ 或 1 ppm Zn2+ 有助於 coenzyme Q3 B (CoQ3B) 還原為 antroquinonol B (AQB) 的反應，造成 AQB 在上游累積而增加 4-AAQB 的生合成。有趣的是，Zn2+ 與 Cu2+ 皆會造成 coenzyme Q3 (CoQ3) 累積，但添加 Zn2+ 的組別卻無法順利將 CoQ3 轉換成 AQ。添加 100 ppm 4-NBA 降低 quinone ring 與 farnesyl diphosphate (FPP) 或 farnesyl diphosphate B (FPPB) 結合形成的中間產物；然而 AQB 與 4-AAQB 卻有累積的現象，顯示可能具有兩種不同的酵素系統分別參與 AQ 與 4-AAQB 的生合成。本研究的實驗結果顯示，牛樟芝菌絲體色素表現與 AQ 及 4-AAQB 生合成途徑沒有直接的關係。目前已知的 AQ 及 4-AAQB 生合成途徑的中間產物，包含 coenzyme Q0 (CoQ0)、CoQ3 等具有顏色的泛醌，並非牛樟芝菌絲體主要的色素成分。

Antrodia cinnamomea is a well-known pharmaceutical fungus native to Taiwan. Among lots of bioactive secondary metabolites contained in the mycelium of A. cinnamomea, the ubiquinone-like compounds, antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), are attractive due to their outstanding anti-cancer effects. In our previous studies, we have observed that the yields of AQ and 4-AAQB are positively correlated to the pigmentation of the mycelium during submerged fermentation of A. cinnamomea. Therefore, this study aims to discuss the relationship between pigmentation and biosynthetic pathway of AQ and 4-AAQB via adding copper ion (Cu2+), zinc ion (Zn2+), and 4-nitrobenzoate (4-NBA) in culture media during submerged fermentation. In pigmentation, 10 ppm Cu2+ caused the redshift of visible spectrum attributed to the composition change; but 1 ppm Zn2+ and 100 ppm 4-NBA decreased the pigmentation, especially the dose-dependent effect of 4-NBA. 10 ppm Cu2+, 1 ppm Zn2+ and 100 ppm 4-NBA could increase the yield of 4-AAQB, but AQ severely inhibited in the presence of 1 ppm Zn2+ and 100 ppm 4-NBA. We use UHPLC-MS/MS to analyze the effects of treatments on the intermediates in the AQ and 4-AAQB biosynthetic pathways. The results show that 10 ppm Cu2+ and 1 ppm Zn2+ would promote the conversion of coenzyme Q3 B (CoQ3B) to antroquinonol B (AQB) and contribute to 4-AAQB accumulation. Interestingly, Both Cu2+ and Zn2+ would lead to coenzyme Q3 (CoQ3) accumulation, however, the conversion of CoQ3 to AQ was inhibited in the presence of Zn2+. 100 ppm 4-NBA reduced the accumulation of intermediates, deriving from quinone ring combined with farnesyl diphosphate (FPP) or farnesyl diphosphate B (FPPB), except AQB and 4-AAQB. This finding suggested that there are two different enzymatic systems involved in the biosynthesis of AQ and 4-AAQB. In this study, we deduce that the pigmentation of A. cinnamomea mycelium is unrelated to the biosynthetic pathway of AQ and 4-AAQB. The intermediates including colored ubiquinones, coenzyme Q0 (CoQ0) and CoQ3, are not the major pigments of A. cinnamomea.