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

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

Abstract

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

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.

Figures/Tables 11

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

Table 1 Primers for random mutation library construction

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

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）

Fig. 3 FACS analysis of negative and positive strains

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

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

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

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

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

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

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

References 25

[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