基于单细胞超高通量筛选的α-1,2-岩藻糖基转移酶定向进化  

Directed Evolution of α-1,2-fucosyltransferase by a Single-cell Ultra-high-throughput Screening Method

作　　者：张雪 谭玉萌 蒋海霞 杨广宇[1] ZHANG Xue;TAN Yu-meng;JIANG Hai-xia;YANG Guang-yu(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240

出　　处：《生物技术通报》2022年第1期289-298,共10页Biotechnology Bulletin

基　　金：国家重点研发计划(2018YFE0200501,2020YFA0907902);国家自然科学基金项目(32030063,21627812)。

摘　　要：2’-岩藻糖基乳糖在婴幼儿配方奶粉、保健品和医药等产品开发方面极具应用价值。幽门螺杆菌(Helicobacter pylori NCTC11639)来源的α-1,2-岩藻糖基转移酶(FutC)是目前合成2’-岩藻糖基乳糖的重要生物催化剂,但是天然酶存在异源表达量低、催化活性差等缺陷。针对α-1,2-岩藻糖基转移酶定向进化过程中缺少高通量筛选方法的问题,发展了基于荧光激活细胞分选(fluorescence-activated cell sorting,FACS)的FutC超高通量筛选方法。对FutC进行了定向进化实验,通过对FutC的随机突变库进行了3轮筛选,从库容量为5.4×10^(5)突变文库中成功筛选出活力提高2.6倍、2.7倍、3倍的3种突变体(K282E,K102E,R105C),证明了此筛选方法的有效性。本研究为α-1,2-岩藻糖基转移酶的分子活性改造奠定了良好基础。2’-fucosyllactose has great application value in the development of infant formula milk powder,health care products and pharmaceutical products.α-1,2-fucosyltransferase(FutC)from Helicobacter pylori NCTC11639 is an important biocatalyst for the synthesis of 2’-fucosyllactose,but it is suffered from poor heterologous expression and low catalytic activity.In this study,an ultra-high-throughput screening method based on fluorescence-activated cell sorting(FACS)of FutC was established,aiming at the problem of lack of screening methods in the directed evolution of α-1,2-fucosyltransferase.Then,directed evolution of α-1,2-fucosyltransferase was carried out.Mutants K282E,K102E,and R105C were successfully obtained after 3 rounds of screening from the 5.4×10^(5) random mutation library,which showed 2.6 times,2.7 times and 3 times higher activities than the wild-type enzyme,thus the effectiveness of this screening method was proved.This study laid a good foundation for the further molecular directed evolution of α-1,2-fucosyltransferase.

关 键 词：岩藻糖基转移酶 定向进化 超高通量筛选方法 催化效率 

分 类 号：Q814[生物学—生物工程]