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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0516

摘要/Abstract

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5.4×10⁵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。

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

Abstract:

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.

Key words: fucosyltransferase, directed evolution, ultra-high-throughput screening method, catalytic activity

张雪, 谭玉萌, 蒋海霞, 杨广宇. 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[J]. 生物技术通报, 2022, 38(1): 289-298.

ZHANG Xue, TAN Yu-meng, JIANG Hai-xia, YANG Guang-yu. Directed Evolution of α-1,2-fucosyltransferase by a Single-cell Ultra-high-throughput Screening Method[J]. Biotechnology Bulletin, 2022, 38(1): 289-298.

图/表 11

图1 荧光底物结构和FACS筛选策略示意图 A：两种偶联了荧光基团的乳糖底物;B：单细胞胞内荧光捕获示意图

Fig. 1 Fluorescent substrate structure and the schematic diagram of FACS screening strategy A：The fluorescent substrates. B：Schematic diagram of single cell intracellular fluorescence capture

表1 随机突变库构建引物设计

Table 1 Primers for random mutation library construction

引物Primer	序列Sequence（5'-3'）
FutC-ep -F	CGAGCTCGACGATGACGATAAAATGGCCTTTAA
FutC-ep- R	CCCAAGCTTGGGTCAATCTAAAGCGTTATAC

图2 TLC分析FutC的荧光产物和紫外灯照射分析阴性菌株和阳性菌株的胞内荧光富集反应 A：TLC分析两种荧光底物（Negtive）以及FutC催化荧光底物生成的荧光产物（FutC）;B：紫外灯照射下的阴性菌株和阳性菌株的胞内荧光富集反应的对比,阴性菌株（Negtive,JM107 Nan- LacZ- pUCKC18-FKP/pUC18）和阳性菌株（FutC,JM107 Nan- LacZ- pUCKC18-FKP/pUC18-FutC）

Fig. 2 TLC analysis of fluorescent products and intra-cellular fluorescence enrichment reaction analysis of negative and positive strains under UV light irradiation A：TLC analysis of the two fluorescent substrates（Negtive）and the fluorescent products catalyzed by FutC（FutC）. B：Comparison of the intracellular fluorescence enrichment reaction of the negative strain and the positive strain under UV light irradiation,the negative strain（Negtive,JM107 Nan- LacZ- pUCKC18-FKP/pUC18）and positive strains（FutC,JM107 Nan- LacZ- pUCKC18-FKP/pUC18-FutC）

图3 阴性菌株和阳性菌株的FACS分析

Fig. 3 FACS analysis of negative and positive strains

图4 细菌复苏率和分选准确率的优化 C：优化前的分选准确率;D：优化后的分选准确率

Fig. 4 Optimization of bacterial recovery rate and sorting accuracy C：Sorting accuracy before optimization. D：Sorting accuracy after optimization

表2 FutC的模式分选

Table 2 Model screening of FutC

分选前阳性比例 Proportion of positive cells before sorting/%	分选后阳性比例 Proportion of positive cells after sorting/%	富集倍数 Enrichment factor
50	100	2
10	100	10
1	90	90
0.1	73.3	733

图5 FutC随机突变文库构建 A：不同 Mn2+浓度条件下futC基因的易错PCR产物,1,2,3,4分别为：0,0.04, 0.06, 0.1 mmol/L Mn2+条件下的PCR产物;B：菌落PCR验证突变文库的连接效率,5为阴性对照

Fig. 5 Construction of the FutC random mutation library A：Error-prone PCR products of futC gene with different Mn2+ concentration,1, 2, 3, 4 are：PCR products with 0, 0.04, 0.06, and 0.1 mmol/L Mn2+. B：Ligation efficiency of random mutation library,and 5 is a negative control

图6 流式细胞仪分析分选前的随机突变文库和分选2轮后的随机突变文库

Fig. 6 FACS analysis of the FutC random mutation libraries before screening and after two rounds of screening

图7 TLC分析复筛得到的单克隆的粗酶液活力

Fig. 7 TLC analysis of monoclonal crude enzyme activity after re-screening WT：Wild type

图8 HPLC酶活测定方法的建立及FutC野生型和突变体的蛋白纯化 A：底物lactose-bodipy和产物2'-fucosyllactose-bodipy的HPLC检测;B：产物2'-fucosyllactose-bodipy的定量标准曲线;C：纯化的FutC野生型和突变体酶蛋白的SDS-PAGE分析,WT：野生型,1：K102E,2：R105C,3：K282E

Fig. 8 Establishment of HPLC enzyme activity determin-ation method and protein purification of FutC WT and mutant A：HPLC analysis of substrate lactose-bodipy and product 2'-fucosyllactose-bodipy. B：Standard curve for the quantification of product 2'-fucosyllactose-bodipy. C：SDS-PAGE analysis of purified FutC wild-type and mutant proteins,WT：wild type,1：K102E, 2：R105C,3：K282E

表3 FutC野生型和突变体对荧光乳糖lactose-bodipy的比活力

Table 3 Specific activities of WT FutC and selected mutants using lactose-bodipy as acceptor

酶Enzyme	突变位点Mutation	比活力Specific activity/（mU·mg^-1）
WT	None	57.6±3.6
1	K102E	156.8±2.9
2	R105C	175.7±11.0
3	K282E	151.9±7.9

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