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

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5.4×10⁵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。

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

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 5.4×10⁵ 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.