Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing

Autores
Ravn, Jonas L.; Manfrão-Netto, João H.C.; Schaubeder, Jana B.; Torello Pianale, Luca; Spirk, Stefan; Ciklic, Ivan Francisco; Geijer, Cecilia
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
EEA Mendoza, INTA
Fil: Ravn, Jonas L. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Manfrão-Netto, João H.C. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Manfrão-Netto, João H.C. Brazilian Biorenewables National Laboratory. Brazilian Center for Research in Energy and Materials (CNPEM); Brasil
Fil: Schaubeder, Jana B. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); Austria
Fil: Torello Pianale, Luca. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Spirk, Stefan. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); Austria
Fil: Ciklic, Ivan F. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Ciklic, Ivan F. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentina
Fil: Geijer, Cecilia. Chalmers University of Technology. Department of Life Sciences; Suecia
Fuente
Microbial Cell Factories 23 : 85. (March 2024)
Materia
Saccharomyces cerevisiae
CRISPR
Fermentation
Yeasts
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas
Fermentación
Levadura
Glucuronoxilano
Levadura
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/19142

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spelling Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editingRavn, Jonas L.Manfrão-Netto, João H.C.Schaubeder, Jana B.Torello Pianale, LucaSpirk, StefanCiklic, Ivan FranciscoGeijer, CeciliaSaccharomyces cerevisiaeCRISPRFermentationYeastsRepeticiones Palindrómicas Cortas Agrupadas y Regularmente InterespaciadasFermentaciónLevaduraGlucuronoxilanoLevaduraBackground: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.EEA Mendoza, INTAFil: Ravn, Jonas L. Chalmers University of Technology. Department of Life Sciences; SueciaFil: Manfrão-Netto, João H.C. Chalmers University of Technology. Department of Life Sciences; SueciaFil: Manfrão-Netto, João H.C. Brazilian Biorenewables National Laboratory. Brazilian Center for Research in Energy and Materials (CNPEM); BrasilFil: Schaubeder, Jana B. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); AustriaFil: Torello Pianale, Luca. Chalmers University of Technology. Department of Life Sciences; SueciaFil: Spirk, Stefan. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); AustriaFil: Ciklic, Ivan F. Chalmers University of Technology. Department of Life Sciences; SueciaFil: Ciklic, Ivan F. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Geijer, Cecilia. Chalmers University of Technology. Department of Life Sciences; SueciaBMC2024-08-28T11:08:09Z2024-08-28T11:08:09Z2024-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/19142https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02361-w1475-2859https://doi.org/10.1186/s12934-024-02361-wMicrobial Cell Factories 23 : 85. (March 2024)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-04T09:50:35Zoai:localhost:20.500.12123/19142instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-04 09:50:36.232INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
title Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
spellingShingle Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
Ravn, Jonas L.
Saccharomyces cerevisiae
CRISPR
Fermentation
Yeasts
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas
Fermentación
Levadura
Glucuronoxilano
Levadura
title_short Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
title_full Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
title_fullStr Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
title_full_unstemmed Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
title_sort Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
dc.creator.none.fl_str_mv Ravn, Jonas L.
Manfrão-Netto, João H.C.
Schaubeder, Jana B.
Torello Pianale, Luca
Spirk, Stefan
Ciklic, Ivan Francisco
Geijer, Cecilia
author Ravn, Jonas L.
author_facet Ravn, Jonas L.
Manfrão-Netto, João H.C.
Schaubeder, Jana B.
Torello Pianale, Luca
Spirk, Stefan
Ciklic, Ivan Francisco
Geijer, Cecilia
author_role author
author2 Manfrão-Netto, João H.C.
Schaubeder, Jana B.
Torello Pianale, Luca
Spirk, Stefan
Ciklic, Ivan Francisco
Geijer, Cecilia
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Saccharomyces cerevisiae
CRISPR
Fermentation
Yeasts
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas
Fermentación
Levadura
Glucuronoxilano
Levadura
topic Saccharomyces cerevisiae
CRISPR
Fermentation
Yeasts
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas
Fermentación
Levadura
Glucuronoxilano
Levadura
dc.description.none.fl_txt_mv Background: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
EEA Mendoza, INTA
Fil: Ravn, Jonas L. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Manfrão-Netto, João H.C. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Manfrão-Netto, João H.C. Brazilian Biorenewables National Laboratory. Brazilian Center for Research in Energy and Materials (CNPEM); Brasil
Fil: Schaubeder, Jana B. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); Austria
Fil: Torello Pianale, Luca. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Spirk, Stefan. Graz University of Technology. Institute of Bioproducts and Paper Technology (BPTI); Austria
Fil: Ciklic, Ivan F. Chalmers University of Technology. Department of Life Sciences; Suecia
Fil: Ciklic, Ivan F. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; Argentina
Fil: Geijer, Cecilia. Chalmers University of Technology. Department of Life Sciences; Suecia
description Background: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. Results: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L− 1 after 48 h under oxygen limited condition in bioreactor fermentations. Conclusion: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast’s expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.
publishDate 2024
dc.date.none.fl_str_mv 2024-08-28T11:08:09Z
2024-08-28T11:08:09Z
2024-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
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info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/19142
https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02361-w
1475-2859
https://doi.org/10.1186/s12934-024-02361-w
url http://hdl.handle.net/20.500.12123/19142
https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-024-02361-w
https://doi.org/10.1186/s12934-024-02361-w
identifier_str_mv 1475-2859
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv BMC
publisher.none.fl_str_mv BMC
dc.source.none.fl_str_mv Microbial Cell Factories 23 : 85. (March 2024)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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