設計並建立以酵素催化合成醣基化黃酮素化合物之平台

Abstract

醣基化(glycosylation)為生物合成中常見的修飾反應，分子的親疏水性、溶解度、生物活 性及構形等會隨之改變。本計畫的主要目的是以枯草桿菌(Bacillus cereus)的大環內酯醣 基轉移酶(macrolide glycosyltransferases，MGT)為主，配合碗豆的蔗糖合成酶(sucrose synthase，SUS)與其它酵素，建立一個兼具高位向專一性及醣基多樣性的類黃酮醣基衍 生物生產平台。其中我們將以槲皮素為模型，針對MGT 的兩個鍵結區--醣基鍵結區與 受質鍵結區--進行突變改造，增加醣基的選擇性和槲皮素醣基化位向的專一性。除了 MGT 的突變與其表現和純化，我們也將建立一套篩檢不同位向專一醣基化突變株的方 法，期望此平台的建立，能對槲皮素及相關類黃酮類的醣基化有更進一步的了解。 第一年至第二年主要工作項目包括： (1)轉糖酵素的活性和產物分析方法 (2)MGT、SUS、輔助酵素的大量表現與純化製程的最適化 (3)突變株篩選方法的建立與測試 (4)利用定點突變/隨機突變法對MGT 醣基鍵結區與受質鍵結區進行改造 第二年至第三年主要工作項目包括： (1)篩選具有高位向專一性與醣基多樣性的突變株，進行大量表現與純化 (2)突變株的活性與產物的分析鑑定 (3)統合MGT、SUS、輔助酵素，建立兼具高位向專一性及醣基多樣性的quercetin 醣基 衍生物的合成平台。

Glycosylation is a common modification process in biosynthesis, and it can change the hydrophobicity, solubility, biological activity and/orstructure conformation of a molecule. The main objective of this project is to build a MGT (macrolide glycosyltransferase)-based platform for the production of regio-selective glycosyl derivativesof flavonoids. Quercetin will be used as a model substrate (acceptor), and protein engineering will used to improve the regio-selectivity of glycosylation and diversity of activated sugars that MGT can utilize. In addition to protein engineering, screening procedures will be established for identifying specific mutants. By doing so, we expect to gain better understanding of the glycosylation of quercetin and flavonoids. The main tasks for the first-half period include the overexpression and purification of MGT, sucrose synthase (SUS), and supporting enzymes, the methodology development of enzymatic assay and product analysis. Protein engineering (site and random mutagenesis) of MGT and methods established for screening desired mutants will also be conducted in this period of time. In the second-half period, the main tasks include searching/overexpression/purification of regio-selective and/or broad sugar-specificity mutants. Combine engineered MGT, SUS, and supporting enzymes to build the proposed platform for enzymatic synthesis of flavonoid glycosides.