Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FU...

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Autores: Moya Gonzálvez, Eva M., Zeuner, Birgitte, Thorhallsson, Albert, Holck, Jesper, Palomino Schätzlein, Martina, Rodríguez-Díaz, Jesús, Meyer, Anne S., Yebra, María Jesús
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2024
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/360657
Acceso en línea:http://hdl.handle.net/10261/360657
https://api.elsevier.com/content/abstract/scopus_id/85193933146
Access Level:acceso abierto
Palabra clave:2′-fucosyllactose
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
id ES_a8d2eafdf1f1960abded90c522587c4a
oai_identifier_str oai:digital.csic.es:10261/360657
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
title Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
spellingShingle Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
Moya Gonzálvez, Eva M.
2′-fucosyllactose
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
title_short Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
title_full Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
title_fullStr Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
title_full_unstemmed Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
title_sort Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome
dc.creator.none.fl_str_mv Moya Gonzálvez, Eva M.
Zeuner, Birgitte
Thorhallsson, Albert
Holck, Jesper
Palomino Schätzlein, Martina
Rodríguez-Díaz, Jesús
Meyer, Anne S.
Yebra, María Jesús
author Moya Gonzálvez, Eva M.
author_facet Moya Gonzálvez, Eva M.
Zeuner, Birgitte
Thorhallsson, Albert
Holck, Jesper
Palomino Schätzlein, Martina
Rodríguez-Díaz, Jesús
Meyer, Anne S.
Yebra, María Jesús
author_role author
author2 Zeuner, Birgitte
Thorhallsson, Albert
Holck, Jesper
Palomino Schätzlein, Martina
Rodríguez-Díaz, Jesús
Meyer, Anne S.
Yebra, María Jesús
author2_role author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia e Innovación (España)
Agencia Estatal de Investigación (España)
Generalitat Valenciana
Moya Gonzálvez, Eva María [0000-0003-4109-5459]
Zeuner, Birgitte [0000-0002-8593-6742]
Thorhallsson, Albert [0000-0002-4003-7647]
Holck, Jesper 0000-0002-8072-1101]
Palomino Schätzlein, Martina [0000-0001-7303-0743]
Rodríguez-Díaz, Jesús [0000-0002-9698-7684]
Meyer, Anne S. [0000-0001-8910-9931]
Yebra, María Jesús [0000-0003-4638-986X]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv 2′-fucosyllactose
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
topic 2′-fucosyllactose
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
3′-fucosyllactose
GH29
Human milk oligosaccharides
Protein engineering
α-L-fucosidase
description Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.
publishDate 2024
dc.date.none.fl_str_mv 2024
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/360657
https://api.elsevier.com/content/abstract/scopus_id/85193933146
url http://hdl.handle.net/10261/360657
https://api.elsevier.com/content/abstract/scopus_id/85193933146
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115403RB-C21
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115403RB-C22
Applied microbiology and biotechnology
The underlying dataset has been published as supplementary material of the article in the publisher platform at https://doi.org/10.1007/s00253-024-13178-3
https://doi.org/10.1007/s00253-024-13178-3
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
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spelling Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenomeMoya Gonzálvez, Eva M.Zeuner, BirgitteThorhallsson, AlbertHolck, JesperPalomino Schätzlein, MartinaRodríguez-Díaz, JesúsMeyer, Anne S.Yebra, María Jesús2′-fucosyllactose3′-fucosyllactoseGH29Human milk oligosaccharidesProtein engineeringα-L-fucosidase3′-fucosyllactoseGH29Human milk oligosaccharidesProtein engineeringα-L-fucosidaseFucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work is part of the Grant PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033. The study was also supported by Valencian Government grant AICO/2021/033. EMM-G was supported by the Grant PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF Investing in your future.” IATA-CSIC is a Center of Excellence Accreditation Severo Ochoa (CEX2021-001189-S MCIN/AEI/10.13039/501100011033).Peer reviewedSpringer NatureMinisterio de Ciencia e Innovación (España)Agencia Estatal de Investigación (España)Generalitat ValencianaMoya Gonzálvez, Eva María [0000-0003-4109-5459]Zeuner, Birgitte [0000-0002-8593-6742]Thorhallsson, Albert [0000-0002-4003-7647]Holck, Jesper 0000-0002-8072-1101]Palomino Schätzlein, Martina [0000-0001-7303-0743]Rodríguez-Díaz, Jesús [0000-0002-9698-7684]Meyer, Anne S. [0000-0001-8910-9931]Yebra, María Jesús [0000-0003-4638-986X]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242024info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/360657https://api.elsevier.com/content/abstract/scopus_id/85193933146reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115403RB-C21info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2020-115403RB-C22Applied microbiology and biotechnologyThe underlying dataset has been published as supplementary material of the article in the publisher platform at https://doi.org/10.1007/s00253-024-13178-3https://doi.org/10.1007/s00253-024-13178-3Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3606572026-05-22T06:33:51Z
score 15,812429

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