Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
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...
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
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BMC
2024
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| Online Access: | 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 |
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| author | Ravn, Jonas L. Manfrão-Netto, João H.C. Schaubeder, Jana B. Torello Pianale, Luca Spirk, Stefan Ciklic, Ivan Francisco Geijer, Cecilia |
| author_browse | Ciklic, Ivan Francisco Geijer, Cecilia Manfrão-Netto, João H.C. Ravn, Jonas L. Schaubeder, Jana B. Spirk, Stefan Torello Pianale, Luca |
| author_facet | Ravn, Jonas L. Manfrão-Netto, João H.C. Schaubeder, Jana B. Torello Pianale, Luca Spirk, Stefan Ciklic, Ivan Francisco Geijer, Cecilia |
| author_sort | Ravn, Jonas L. |
| collection | INTA Digital |
| 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. |
| format | info:ar-repo/semantics/artículo |
| id | INTA19142 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | BMC |
| publisherStr | BMC |
| record_format | dspace |
| spelling | INTA191422024-08-28T11:31:14Z Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing Ravn, Jonas L. Manfrão-Netto, João H.C. Schaubeder, Jana B. Torello Pianale, Luca Spirk, Stefan Ciklic, Ivan Francisco Geijer, Cecilia Saccharomyces cerevisiae CRISPR Fermentation Yeasts Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Fermentación Levadura Glucuronoxilano Levadura 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 2024-08-28T11:08:09Z 2024-08-28T11:08:09Z 2024-03 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng 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) application/pdf BMC Microbial Cell Factories 23 : 85. (March 2024) |
| spellingShingle | Saccharomyces cerevisiae CRISPR Fermentation Yeasts Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Fermentación Levadura Glucuronoxilano Levadura Ravn, Jonas L. Manfrão-Netto, João H.C. Schaubeder, Jana B. Torello Pianale, Luca Spirk, Stefan Ciklic, Ivan Francisco Geijer, Cecilia 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 |
| 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_short | 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 |
| topic | Saccharomyces cerevisiae CRISPR Fermentation Yeasts Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Fermentación Levadura Glucuronoxilano Levadura |
| 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 |
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