改造大肠杆菌合成海藻糖途径以高效合成海藻糖

Construction of a recombinant Escherichia coli for high trehalose production

摘要:

海藻糖是相容性溶质的一种，因其具有多种生物学功能，在食品、化妆品、药品以及器官移植等方面均有很广泛应用。然而近几年生产海藻糖主要集中在使用酶催化的方法，虽然这种方法的转化效率高，但是却存在着副产物的问题，难以得到高纯度的海藻糖产品，严重制约了海藻糖的应用。本文通过基因工程技术在大肠杆菌Escherichia coli中构建了海藻糖高效合成新途径，通过全细胞催化合成海藻糖。利用PCR技术在哈氏噬纤维菌Cytophaga hutchinsonii中克隆获得海藻糖双功能合成酶基因 (tpsp)，采用E. coli pTac-HisA高效表达载体，实现海藻糖双功能合成酶基因 (tpsp) 高效表达，利用高效表达菌株进行全细胞催化，将葡萄糖高效转化为海藻糖。结果表明C. hutchinsonii海藻糖合成酶基因 (tpsp) 在E. coli中成功实现表达，该酶能够在胞内将葡萄糖高效转化为海藻糖，并将其转运到胞外，实现海藻糖的高效率合成，海藻糖的产量提高到1.2 g/L，相对转化率为21%。当将此高产菌株在发酵罐中进行转化时，海藻糖的产量达到13.3 g/L，葡萄糖的相对转化率达到48.6%。采用C. hutchinsonii海藻糖合成酶基因高效表达并且应用于海藻糖全细胞合成催化在国内外尚属首次报道，海藻糖的转化率及产率都已达到文献报道最高水平，本研究为开拓海藻糖生产新技术奠定了基础。

Abstract:

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and low purity. In this study, high amount of trehalose was produced by recombinant E. coli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinsonii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonii has been successfully expressed in E. coli. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.