利用釀酒酵母菌生合成葫蘆素B前驅物

Abstract

葫蘆素B是葫蘆科植物的次級代謝物，具有廣泛的藥理特性。但該化合物在植物中含量低且結構複雜，難以利用化學合成或溶劑萃取獲得。因此，利用釀酒酵母生物合成葫蘆素B可做為一種替代方法。先前實驗室研究，已透過酵母菌成功將2,3-氧化角鯊烯導向葫蘆二烯醇的合成，作為葫蘆素B的前驅物，然需進一步進行羥基化等修飾。過去文獻顯示植物之細胞色素P450酶與細胞色素還原酶共同作用時，可修飾葫蘆二烯醇。因此，本研究選擇不同植物來源之酵素，並將相關基因整合到酵母中，希望藉由基因工程建構合成葫蘆素B前驅物之酵母菌。實驗中將羅漢果葫蘆二烯醇酶SgCS、苦瓜細胞色素還原酶McCPR及香瓜細胞色素P450酶Cm890基因整合至酵母菌中，以西方墨點法結果顯示酵母菌成功表現蛋白，而且插入外源基因並未影響菌株的生長，並且結果顯示酵母菌細胞可生成化合物11-carbonylcucurbitadienol和11-carbonyl-20β-hydroxycucurbitadienol。為提升這些化合物的含量，以利後續葫蘆素B的合成，研究進一步修改麥角固醇途徑中的HMG1基因表現量，以期增加關鍵前驅物2,3-氧化角鯊烯的含量，然而並未成功表達此基因於酵母菌中，同時會造成原先整合之基因被剔除。因此，未來研究可進一步選用誘導型啟動子調整基因之表現，以達成酵母菌中葫蘆素B的建構。

Cucurbitacin B is a secondary metabolite produced by Cucurbitaceae plants with various pharmacological properties. Due to the low contents and complexity of the structure of this compound in plants, it is difficult to be synthesized chemically or extracted by solvents. Therefore, biosynthesis using yeast, Saccharomyces cerevisiae, can be an alternative method to obtain cucurbitacin B. In our previous study, we modified the yeast and successfully produced cucurbitadienol, the precursor of cucurbitacin B. However, further hydroxylation is required for the synthesis of cucurbitacin B. According to studies, plant cytochrome P450s (CYPs) are often co-expressed with cytochrome P450s reductase (CPR) to chemically modify cucurbitadienol. Thus, in the study, enzymes will be selected from various plants, and the related genes will be integrated into yeast. This process is designed to enable the biosynthesis of cucurbitacin B precursors within the yeast. Our results showed that the recombinant yeast strains with cucurbitadienol synthase from Siraitia grosvenorii, CPR from Momordica charantia and Cm890 from C. melo successfully expressed these proteins. The spotting assay revealed that overexpressing these genes did not impact cell growth, and the yeast produced minor quantities of 11-carbonylcucurbitadienol and 11-carbonyl-20β-hydroxycucurbitadienol. To enhance the synthesis of these compounds, increasing the content of the precursor 2,3-oxidosqualene is essential. This was attempted through the modification of the HMG1 gene in the ergosterol pathway, but the data revealed that this gene was not successfully integrated into the yeast, leading to the loss of previously integrated genes. In the future, the selection of inducible promoters may be further explored to regulate the expression of these genes, with the aim of achieving the synthesis of cucurbitacin B in yeast.