生物技术通报 ›› 2022, Vol. 38 ›› Issue (1): 289-298.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0516
收稿日期:
2021-04-15
出版日期:
2022-01-26
发布日期:
2022-02-22
作者简介:
张雪,女,硕士研究生,研究方向:糖基转移酶的定向进化;E-mail: 基金资助:
ZHANG Xue(), TAN Yu-meng, JIANG Hai-xia, YANG Guang-yu()
Received:
2021-04-15
Published:
2022-01-26
Online:
2022-02-22
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。
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
引物Primer | 序列Sequence(5'-3') |
---|---|
FutC-ep -F | CGAGCTCGACGATGACGATAAAATGGCCTTTAA |
FutC-ep- R | CCCAAGCTTGGGTCAATCTAAAGCGTTATAC |
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
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 |
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
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 |
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 |
[1] |
Tu Z, Lin YN, Lin CH. Development of fucosyltransferase and fucosidase inhibitors[J]. Chem Soc Rev, 2013, 42(10):4459-4475.
doi: 10.1039/c3cs60056d URL |
[2] |
Keeley TS, Yang SY, Lau E. The diverse contributions of fucose linkages in cancer[J]. Cancers, 2019, 11(9):1241.
doi: 10.3390/cancers11091241 URL |
[3] |
Thomas D, Rathinavel AK, Radhakrishnan P. Altered glycosylation in cancer:a promising target for biomarkers and therapeutics[J]. Biochim Biophys Acta Rev Cancer, 2021, 1875(1):188464.
doi: 10.1016/j.bbcan.2020.188464 URL |
[4] |
Danishefsky SJ, Shue YK, Chang MN, et al. Development of Globo-H cancer vaccine[J]. Acc Chem Res, 2015, 48(3):643-652.
doi: 10.1021/ar5004187 URL |
[5] |
Huang CS, Yu AL, Tseng LM, et al. Globo H-KLH vaccine adagloxad simolenin(OBI-822)/OBI-821 in patients with metastatic breast cancer:phase II randomized, placebo-controlled study[J]. J Immunother Cancer, 2020, 8(2):e000342.
doi: 10.1136/jitc-2019-000342 URL |
[6] |
Guo J, Jiang W, Li Q, et al. Comparative immunological studies of tumor-associated Lewis X, Lewis Y, and KH-1 antigens[J]. Carbohydr Res, 2020, 492:107999.
doi: 10.1016/j.carres.2020.107999 URL |
[7] |
Bode L. Human milk oligosaccharides:every baby needs a sugar Mama[J]. Glycobiology, 2012, 22(9):1147-1162.
doi: 10.1093/glycob/cws074 URL |
[8] |
He Y, Liu S, Kling DE, et al. The human milk oligosaccharide 2'-fucosyllactose modulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation[J]. Gut, 2016, 65(1):33-46.
doi: 10.1136/gutjnl-2014-307544 URL |
[9] |
Orczyk-Pawiłowicz M, Lis-Kuberka J. The impact of dietary fucosylated oligosaccharides and glycoproteins of human milk on infant well-being[J]. Nutrients, 2020, 12(4):1105.
doi: 10.3390/nu12041105 URL |
[10] | Sprenger N, Binia A, Austin S. Human milk oligosaccharides:factors affecting their composition and their physiological significance[J]. Nestle Nutr Inst Work Ser, 2019, 90:43-56. |
[11] |
Goehring KC, Marriage BJ, Oliver JS, et al. Similar to those who are breastfed, infants fed a formula containing 2'-fucosyllactose have lower inflammatory cytokines in a randomized controlled trial[J]. J Nutr, 2016, 146(12):2559-2566.
pmid: 27798337 |
[12] |
Musilova S, Rada V, Vlkova E, et al. Beneficial effects of human milk oligosaccharides on gut microbiota[J]. Benef Microbes, 2014, 5(3):273-283.
doi: 10.3920/BM2013.0080 pmid: 24913838 |
[13] | Zhu Y, Wan L, Li W, et al. Recent advances on 2'-fucosyllactose:physiological properties, applications, and production approaches[J]. Crit Rev Food Sci Nutr, 2020:1-10. |
[14] |
Zhou WT, Jiang H, Wang LL, et al. Biotechnological production of 2'-fucosyllactose:a prevalent fucosylated human milk oligosaccharide[J]. ACS Synth Biol, 2021, 10(3):447-458.
doi: 10.1021/acssynbio.0c00645 URL |
[15] |
Drouillard S, Driguez H, Samain E. Large-scale synjournal of H-antigen oligosaccharides by expressing Helicobacter pylori alpha1, 2-fucosyltransferase in metabolically engineered Escherichia coli cells[J]. Angew Chem Int Ed Engl, 2006, 45(11):1778-1780.
doi: 10.1002/(ISSN)1521-3773 URL |
[16] |
Chin YW, Kim JY, Lee WH, et al. Enhanced production of 2'-fucosyllactose in engineered Escherichia coli BL21star(DE3)by modulation of lactose metabolism and fucosyltransferase[J]. J Biotechnol, 2015, 210:107-115.
doi: 10.1016/j.jbiotec.2015.06.431 URL |
[17] |
Huang D, Yang K, Liu J, et al. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement[J]. Metab Eng, 2017, 41:23-38.
doi: S1096-7176(16)30233-6 pmid: 28286292 |
[18] |
Arnold FH, Volkov AA. Directed evolution of biocatalysts[J]. Curr Opin Chem Biol, 1999, 3(1):54-59.
pmid: 10021399 |
[19] |
Turner NJ. Directed evolution drives the next generation of biocatalysts[J]. Nat Chem Biol, 2009, 5(8):567-573.
doi: 10.1038/nchembio.203 URL |
[20] |
Aharoni A, Thieme K, Chiu CP, et al. High-throughput screening methodology for the directed evolution of glycosyltransferases[J]. Nat Methods, 2006, 3(8):609-614.
pmid: 16862135 |
[21] |
Yang GY, Rich JR, Gilbert M, et al. Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases[J]. J Am Chem Soc, 2010, 132(30):10570-10577.
doi: 10.1021/ja104167y URL |
[22] |
Tan YM, Zhang Y, Han YB, et al. Directed evolution of an α 1, 3-fucosyltransferase using a single-cell ultrahigh-throughput screening method[J]. Sci Adv, 2019, 5(10):eaaw8451. DOI: 10.1126/sciadv.aaw8451.
doi: 10.1126/sciadv.aaw8451 |
[23] |
Stein D, Lin YN, Lin CH. Characterization of Helicobacter pylori α1, 2-fucosyltransferase for enzymatic synjournal of tumor-associated antigens[J]. Adv Synth Catal, 2008, 350(14/15):2313-2321.
doi: 10.1002/adsc.v350:14/15 URL |
[24] |
Engels L, Elling L. WbgL:a novel bacterial α1, 2-fucosyltransferase for the synjournal of 2'-fucosyllactose[J]. Glycobiology, 2014, 24(2):170-178.
doi: 10.1093/glycob/cwt096 URL |
[25] |
Li C, Wu M, Gao X, et al. Efficient biosynjournal of 2'-fucosyllactose using an in vitro multienzyme cascade[J]. J Agric Food Chem, 2020, 68(39):10763-10771.
doi: 10.1021/acs.jafc.0c04221 URL |
[1] | 曲戈, 孙周通. 催化混杂性驱动的酶功能重塑[J]. 生物技术通报, 2023, 39(4): 1-9. |
[2] | 王慕镪, 陈琦, 马薇, 李春秀, 欧阳鹏飞, 许建和. 机器学习方法在酶定向进化中的应用进展[J]. 生物技术通报, 2023, 39(4): 38-48. |
[3] | 郝俊尧, 马富强, 杨广宇. 产碱杆菌Alcaligenes sp.KS-85来源肌酸酶活性中心的关键氨基酸功能研究[J]. 生物技术通报, 2021, 37(3): 75-83. |
[4] | 陈春, 宿玲恰, 夏伟, 吴敬. 定向进化提高来源于Arthrobacter ramosus 的MTHase的热稳定性[J]. 生物技术通报, 2021, 37(3): 84-91. |
[5] | 邱锦, 黄火清, 姚斌, 罗会颖. 解淀粉芽孢杆菌淀粉酶催化活力改良及其在枯草芽孢杆菌中的高效表达[J]. 生物技术通报, 2019, 35(9): 134-143. |
[6] | 任天雷, 杨海泉, 许菲. 基于分子结构与生物信息学等多维度特征的定向进化改造甲基对硫磷水解酶[J]. 生物技术通报, 2018, 34(10): 194-200. |
[7] | 王晓璐, 王钰,刘娇,郑平,路福平. 利用定向进化提高基因工程大肠杆菌的甲醇利用能力[J]. 生物技术通报, 2017, 33(9): 101-109. |
[8] | 郭园, 赵仲麟. 微生物系统定向进化与合成生物学应用研究进展[J]. 生物技术通报, 2017, 33(1): 76-82. |
[9] | 张雪玲,陈小利,李荷. 漆酶Lac1338的酶学特性测定及定向突变对其酶解染料影响[J]. 生物技术通报, 2016, 32(7): 170-177. |
[10] | 吕永坤,堵国成,陈坚,周景文. 合成生物学技术研究进展[J]. 生物技术通报, 2015, 31(4): 134-148. |
[11] | 刘瑜,李丕武. 黑曲霉葡萄糖氧化酶高产基因工程菌研究进展[J]. 生物技术通报, 2013, 0(7): 12-19. |
[12] | 邵敏, 李长福, 葛正龙, 周鹤峰. 基于易错PCR技术定向进化枯草芽孢杆菌β-葡聚糖酶[J]. 生物技术通报, 2013, 0(12): 141-145. |
[13] | 付畅;林海龙;李海莹;王得江;. 蛋白质体外定向进化策略研究进展[J]. , 2012, 0(07): 36-40. |
[14] | 陈英;朱绮霞;张搏;陈东;黄日波;. 基于易错PCR技术的黏质沙雷氏菌脂肪酶基因LipA的定向进化[J]. , 2011, 0(04): 181-185. |
[15] | 贾红红;李玉;刘逸寒;王尧;梁晓梅;路福平;. α1,2-岩藻糖基转移酶基因的克隆及在大肠杆菌中的表达[J]. , 2011, 0(03): 185-190. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||