調控GPP與FPP以探討牛樟芝液態發酵時生合成4-AAQB的機制

Abstract

牛樟芝（Antrodia cinnamomea BCRC 35716）子實體生長於台灣高山的牛樟樹腐木上，為台灣特有的藥用真菌，許多文獻證實其次級代謝物具有多種生理功效，其中又以保肝功效最為著名。然而，樟芝子實體之商業化大量生產不易，因此近年產學界研究轉向生長快速的菌絲體。研究顯示，樟芝菌絲體萃取物抑制肝癌活性不亞於子實體，又以泛醌類衍生物：4-acetylantroquinonol B （4-AAQB）及Antroquinonol（AQ）最具功效，而4-AAQB 對於肝癌細胞的IC50 僅0.1 μg/mL，比AQ 更低，因此，確立4-AAQB生合成途徑為本次實驗目的。 牛樟芝產4-AAQB 的生合成途徑包含結構中與benzoquinone 相關的Polyketide 路徑和與isoprenoid chain 相關的Mevalonate 路徑。Polyketide路徑所產生的benzoquinone 為AQ 與4-AAQB 的共同前驅物。目前已知，farnesyl di-phosphate（FPP）以及farnesyl diphosphate B（FPPB）會競爭benzoquinone 上相同的結合位，最後生合成AQ以及4-AAQB。然而FPPB 之生合成方式尚未明確。本研究提出假說：FPPB 並不是由十五碳的FPP 在末端經γ-lactone 修飾而成，而是藉由十碳的geranyl diphosphate（GPP） 接上一五碳的 isopentenyl diphosphate B （IPPB） 所形成。因此，以兩種手段間接證實此假說，一為添加不同碳鏈長度的isoprenoid chain，一為刺激Mevalonate路徑中生成FPP 的farnesyl diphosphate synthase（FPPS）之酵素活性。透過添加geraniol 及farnesol 作為GPP 及FPP 前驅物，發現在第零天添加geraniol可增加4-AAQB 產量而不增加菌絲體乾重。然而，添加farnesol 只增加菌絲體乾重，說明GPP為FPPB前驅物，而FPP在初期會先被用於生成初級代謝產物。在第六天添加等量的geraniol 及farnesol，發現4-AAQB及AQ皆提升，而添加geraniol又可以更顯著提升4-AAQB 產量。接著以UPLC-MS/MS分析 4-AAQB 及 AQ 生合成途徑之中間產物，發現在添加前驅物的情況下，Polyketide路徑上的中間產物產量皆會提升並累積在上游。其中，又以添加geraniol 會明顯促進4-AAQB 及其上游產物產量，添加farnesol 則是明顯的提升AQ 及其上游產物產量，說明GPP確實為4-AAQB的前驅物，而FPP 是AQ 的前驅物。另一實驗則以加鎂離子刺激FPPS 的酵素活性時，結果發現4-AAQB 產量下降，推測FPPS活性上升時，刺激了isopentenyl diphosphate（IPP）及GPP轉為FPP，減少GPP轉化成FPPB。上述研究證實4-AAQB 上之FPPB 與FPP 為競爭關係非上下游關係，證實假說成立。 Mevalonate 路徑為生物體合成異戊二烯結構的重要途徑，會利用acetyl-CoA 代謝成五碳的雙磷酸結構IPP，並以IPP 為單元結構，透過頭尾相接的方式來增長碳鏈結構。基於上述所推測之FPPB 的生成途徑，形成雙磷酸結構為其必要步驟。因此，本實驗探討刺激磷酸化作用對代謝活性的影響。結果顯示，添加磷酸根確實可提高4-AAQB 生成量，與預期相符。本研究結果使我們進一步了解4-AAQB 的生合成途徑，同時也能夠應用於4-AAQB之產量的增加。

Antrodia cinnamomea BCRC 35716（AC）is an unique medical fungus originally grows on the rotten wood of Cinnamomum kanehirai in Taiwan. AC possesses a varity of bioactive secondary metabolites and is well known for its anti-hepatocarcinoma function. However, the growth rate of AC fruiting body is slow, thus cultivation of mycelium by submerged fermentation is now a common practice for the production of bioactive compounds. The major anti- hepatocarcinoma bioactive compounds of AC mycelium are 4-acetylantroquinonol B (4-AAQB) and antroquinonol (AQ), and it has been demonstrated the 4-AAQB is more potent in inhibiting the proliferation of hepa-tocellular carcinoma cells than that of AQ. Our laboratory has studied the biosynthetic pathways of AQ and 4-AAQB for sev-eral years and tried to increase the production of 4-AAQB. Previous studies conducted in our lab has already demonstrated that polyketide pathway and mevalonate pathway are associated with the biosynthesis of 4-AAQB in AC. In polyketide pathway, farnesyl diphosphate (FPP) and farnesyl diphosphate B (FPPB) would compete their common precursor, benzoquinone, to biosynthesize AQ and 4-AAQB. We already know that FPP is produced via mevalonate pathway, but the pathway for FPPB synthesis is still not clear. In order to increase the yields of 4-AAQB, we need to investigate the biosyn-thetic relations of FPP and FPPB. This study proposed a hypothesis：FPP cannot convert to FPPB directly. We as-sumed that FPPB is formed by the binding of GPP with IPPB. We examined this hy-pothesis indirectly by two methods, one was the addition of isoprenoid chains with dif-ferent carbon chain lengths, and the other was to activate farnesyl diphosphate synthase to promote FPP production. First, we added geraniol and farnesol as analogs, and found that addition of geraniol at the early stage of fermentation could increase 4-AAQB production without increasing biomass. However, addition of farnesol only increased biomass. These results indicated that GPP is a precursor of FPPB, and FPP can only be used to generate primary metabolites. In the next experiment, we added the same amount of geraniol and farnesol on the sixth day during fermentation. The results showed that both 4-AAQB and AQ production were improved, and the addition of ge-raniol could significantly increase 4-AAQB production. Then, UPLC-MS/MS was used to detect the intermediates of biosynthetic pathway of 4-AAQB and AQ. Experimental results revealed that addition of precursors, the upstream compounds accumulated in the polyketide pathway and most of intermediates production were increased. As men-tioned above, geraniol can increase 4-AAQB and its intermediates production, and farnesol can increase AQ and its intermediates production. We speculated that GPP is a precursor of FPPB. Farnesol is a precursor of AQ, and it can also be used for primary metabolism. In the another experiment, we added magnesium ion to stimulates the ac-tivity of FPP synthase, a key enzyme in mevalonate pathway that catalysis the for-mation of FPP. We found that addition of magnesium ion decreased the production of 4-AAQB. This result indicated that the increase of FPP synthase activity would stimu-late the conversion of isopentenyl diphosphate (IPP) and GPP to FPP, and reduce the conversion of GPP to FPPB. These results further confirmed that FPPB and FPP are competing their common precursor, benzoquinone, to synthesize AQ and 4-AAQB. Mevalonate pathway is an important way to synthesize terpenes. Acetyl-CoA is the key precursor in the mevalonate pathway to synthesize IPP and dimethylallyl di-phosphate (DMAPP). Due to the bisphosphonate, each isoprenoid unit can connect and extend to become terpenes. Thus, we thought that stimulation of phosphorylation might be able to increase the production of 4-AAQB. Indeed, the results showed that the ad-dition of phosphate during fermentation can increase 4-AAQB production. Based on these experiments, we gained more understanding about the relationship between FPP and FPPB. Thus, we are able to manipulate the submerged fermentation of Antrodia cinnamomea more easily to increase the yield of 4-AAQB.