Molecular cloning, protein expression and activity assay of triterpenoid tailoring glucosyltransferases from Medicago truncatula, Arabidopsis thaliana and Streptomyces antibioticus

• Main Authors: Chen, Yi-Ju, 陳奕汝
• Other Authors: Wu, Tung-Kung
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/11611874909747703372

碩士 === 國立交通大學 === 生物科技系所 === 101 === Saponins are a group of natural products that are widely distributed in living organisms and possess a large variety of bioactivities, including antimicrobial activity, antifungal activity, anticholesterolemic activity, hemolytic activity, antitumor activity, as well as to being adjuvant to enhance immunity. The biological activities of triterpene saponins are attributed to their structural diversity, composed of various sapogenin and glycone moieties, and can be determined by three steps of biosynthesis: (1) cyclization of oxidosqualene into sapogenin backbone; (2) proton transformation, hydroxylation, or oxidation at different carbon atoms within the structure of sapogenin; and (3) glycosylation reaction to decorate sugar moieties at some hydoxyl groups. Glucosyltransferases (GTs) play a major role in turning inactive compounds into active triterpene saponins. Glycosylation not only enhances the solubility and stability of triterpene saponins but also prolongs their storage in organisms. Based on our previous investigations, Saccharomyces cerevisiae ERG7 is applied to examine structure-functional relationships. Diverse truncated and rearranged unnatural triterpene products are generated and isolated to proceed with further studies on acquiring new bioactivities via GTs-mediated sugar-decoration. In this thesis, four GTs are successfully cloned and expressed in E. coli system, including UGT71G1 (Medicago truncatula), UGT73K1 (Medicago truncatula), UGT80A2 (Arabidopsis thaliana), and OleDA242V/S132F/P67T (Streptomyces antibioticus). Three GTs (UGT71G1, UGT73K1 and OleD-ASP) were successfully purified by nickel affinity chromatography. The activities of these three GTs against nine compounds were screened with TLC and further characterized by HPLC/ESI-MS and ESI-MS/MS. UGT71G1 exhibit catalytic abilities toward hederagenin, -estradiol and one unknown structure compound X, while UGT73K1 catalyzes glucose transfer only to hederagenin. OleD-ASP with boardest substrates capacity that generate glycoside products from hederagenin, beta-estradiol, cis-androsterone, trans-androsterone and compound-X. In the future, products from the above GTs-mediated reactions with transferred glucose moiety will be characterized for various bioactivities, while triterpenes isolated from (ERG7) mutants will be subjected to glycosyltransferases tailoring reactions.