Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing
Abstract 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...
| Principais autores: | , , , , , , |
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| Formato: | Artigo |
| Idioma: | English |
| Publicado em: |
BMC
2024-03-01
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| coleção: | Microbial Cell Factories |
| Assuntos: | |
| Acesso em linha: | https://doi.org/10.1186/s12934-024-02361-w |
| _version_ | 1827315647408766976 |
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| author | Jonas L. Ravn João H.C. Manfrão-Netto Jana B. Schaubeder Luca Torello Pianale Stefan Spirk Iván F. Ciklic Cecilia Geijer |
| author_facet | Jonas L. Ravn João H.C. Manfrão-Netto Jana B. Schaubeder Luca Torello Pianale Stefan Spirk Iván F. Ciklic Cecilia Geijer |
| author_sort | Jonas L. Ravn |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-24T23:02:03Z |
| format | Article |
| id | doaj.art-e1cf28d0323647eabf660487bd4ff520 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-24T23:02:03Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-e1cf28d0323647eabf660487bd4ff5202024-03-17T12:42:53ZengBMCMicrobial Cell Factories1475-28592024-03-0123111410.1186/s12934-024-02361-wEngineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editingJonas L. Ravn0João H.C. Manfrão-Netto1Jana B. Schaubeder2Luca Torello Pianale3Stefan Spirk4Iván F. Ciklic5Cecilia Geijer6Department of Life Sciences, Chalmers University of TechnologyDepartment of Life Sciences, Chalmers University of TechnologyInstitute of Bioproducts and Paper Technology (BPTI), Graz University of TechnologyDepartment of Life Sciences, Chalmers University of TechnologyInstitute of Bioproducts and Paper Technology (BPTI), Graz University of TechnologyDepartment of Life Sciences, Chalmers University of TechnologyDepartment of Life Sciences, Chalmers University of TechnologyAbstract 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.https://doi.org/10.1186/s12934-024-02361-wYeastXylanXylanaseα-glucuronidaseMetabolic engineeringCo-culture |
| spellingShingle | Jonas L. Ravn João H.C. Manfrão-Netto Jana B. Schaubeder Luca Torello Pianale Stefan Spirk Iván F. Ciklic Cecilia Geijer Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing Microbial Cell Factories Yeast Xylan Xylanase α-glucuronidase Metabolic engineering Co-culture |
| 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 | Yeast Xylan Xylanase α-glucuronidase Metabolic engineering Co-culture |
| url | https://doi.org/10.1186/s12934-024-02361-w |
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