以酵母菌基因工程增加三萜皂苷前驅物

三萜皂苷(triterpenoid saponins)是植物的次級代謝物之一。在植物中佔有的比例較低、結構複雜，且不易從植物中進行萃取或以化學合成，因此許多研究以生物合成方式產生三萜皂苷。三萜皂苷的結構由皂苷元(aglycon)及糖基以醣苷鍵鍵結組成，總類繁多，羅漢果皂苷(mogrosides)是一種從羅漢果(Siraitia grosvenirii)果實中分離出來的四環三萜皂苷(tetracyclic triterpene saponins)。三萜皂苷的生合成路徑前端為甲羥戊酸途徑(Mevalonate pathway)，此路徑為所有真核細胞生成固醇或三萜化合物均須具備的路徑，因此希望藉由基因工程來改良Saccharomyces cerevisiae原生的麥角固醇(ergosterol)生成路徑，建構出可生合成三萜皂苷的菌株。首先，我們嘗試置換特定基因(HMG1、ERG1、ERG9)的啟動子來調節三萜皂苷非環狀前驅物合成相關的蛋白表現。實驗結果顯示置換HMG1、ERG1的啟動子確實能使細胞內的squalene累積，相較於WT上升了約12倍，且以點試驗證實啟動子置換不會影響到細胞生長，但在該階段的所有菌株中均未看到2,3-oxidosqualene、2,3,22,23-dioxidosqualene累積。推測可能是因為lanosterol synthase活性太強，將整個代謝路徑快速帶往生成麥角固醇。因此後續實驗會建構ERG27基因剔除及upc2-1突變株來阻礙麥角固醇生成路徑，希望最後能如期看到2,3-oxidosqualene、2,3,22,23-dioxidosqualene累積。此實驗產生的菌株亦可做為未來三萜皂苷生成的基本平台。

關鍵字

三萜皂苷；啟動子置換；固醇代謝突變株； Saccharomyces cerevisiae

並列摘要

Triterpenoid saponins are a type of secondary metabolites in plants. Due to the structure complexity and low levels of triterpenoid saponins in plants, it’s difficult extract or synthesize chemically. Recently, biosynthesis of triterpenoid saponins has been applied using biotechnology engineering in microbes. The complexity of triterpenoid saponins depend on the variation of lipophilic aglycon structure and the position and number of glycosides combining to the aglycon. Mogrosides are tetracyclic triterpenoid saponins isolated from the fruits of Siraitia grosvenirii. The biosynthetic pathway of mogrosides is derived from mevalonate pathway presenting in all eukaryotic cells to synthesize sterols or triterpene compounds. The goal of this study is to modify Saccharomyces cerevisiae ergosterol pathway by genetic engineering and to construct the strains that have ability to synthesize triterpenoid saponins. Firstly, the replacement of promoters of HMG1, ERG1 and ERG9 was opted to mediate protein expression related to triterpenoid saponin noncyclic precursors’ synthetic pathway. The data showed that HMG1 and ERG1 promoter replacement would accumulated the amounts of squalene, up to 12-fold increase compared to those in WT. No significant effect on cell growth of the genetic engineered strains. However, the accumulation of 2,3-oxidosqualene or 2,3,22,23-dioxidosqualene could not be observed in all genetic engineered strains. This may be due to that the activity of lanosterol synthase was intense, and lead to the synthesis of lanosterol and ergosteorl, but not the accumulation of 2,3-oxidosqualene or 2,3,22,23-dioxidosqualene. Currently, attempts to establish erg27 knockout and upc2-1 mutant strains may block the metabolic flux to ergosterol pathway. The established platform could possibly promote the biosynthesis of triterpenoids in the future.