Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae
Laccases are enzymes capable of oxidizing phenolic compounds and are important tools in different industrial processes. Heterologous expression of laccases is of great interest in biotechnological applications but achieving high expression levels is challenging. Three different laccases have been id...
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Taylor & Francis Group
2025-01-01
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| Series: | Mycobiology |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/12298093.2024.2439646 |
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| author | Kum-Kang So Fatima Alejandra Hernandez Alvarado Gui-Hwan Han Jeong-Won Kim Tae-Geum Kim Dae-Hyuk Kim |
| author_facet | Kum-Kang So Fatima Alejandra Hernandez Alvarado Gui-Hwan Han Jeong-Won Kim Tae-Geum Kim Dae-Hyuk Kim |
| author_sort | Kum-Kang So |
| collection | DOAJ |
| description | Laccases are enzymes capable of oxidizing phenolic compounds and are important tools in different industrial processes. Heterologous expression of laccases is of great interest in biotechnological applications but achieving high expression levels is challenging. Three different laccases have been identified in the chestnut blight fungus Cryphonectria parasitica, among which a tannic acid-inducible laccase (laccase3) was successfully expressed using Saccharomyces cerevisiae. To obtain high and stable expression of fungal laccases, we cloned the gene encoding an extracellular laccase (Laccase1) of C. parasitica into a yeast episomal vector, used the resulting vectors to transform S. cerevisiae, and optimized the culture conditions of the selected transformants for Laccase1 production. We also tested the significance of the signal peptide of Laccase1 in the secretion of expressed Laccase1 and compared it with the widely used rice amylase signal peptide. Among the four constructs tested using a yeast episomal vector, full-length Laccase1 containing an endogenous signal peptide, showed the highest laccase activity. Interestingly, the stability of the recombinant vector expressing laccase was lower than that of the mock transformant, suggesting a detrimental effect of the Laccase1-expressing vector on host cells. Thus, we optimized the culture conditions to produce Laccase1 and the resulting optimum culture conditions identified through one-factor-at-a -time (OFAT) were 2% sucrose; 3% yeast nitrogen base without amino acid; pH 5.0; and 30 °C. The laccase activity was found to be 2.2 U/mL in optimal culture conditions, resulting in a 6.5-fold increase compared to the conventional culture medium. |
| format | Article |
| id | doaj-art-14e7934485064b2d8f4c0a502eadadd5 |
| institution | Kabale University |
| issn | 1229-8093 2092-9323 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Mycobiology |
| spelling | doaj-art-14e7934485064b2d8f4c0a502eadadd52025-01-29T05:27:35ZengTaylor & Francis GroupMycobiology1229-80932092-93232025-01-01531364610.1080/12298093.2024.2439646Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiaeKum-Kang So0Fatima Alejandra Hernandez Alvarado1Gui-Hwan Han2Jeong-Won Kim3Tae-Geum Kim4Dae-Hyuk Kim5Institute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju, Republic of KoreaDepartment of Molecular Biology, Jeonbuk National University, Jeonju, Republic of KoreaCenter for Industrialization of Agricultural and Livestock Microorganisms, Jeongeup, Republic of KoreaCenter for Industrialization of Agricultural and Livestock Microorganisms, Jeongeup, Republic of KoreaDepartment of Bio-Convergence Science, Jeonbuk National University, Jeongeup, Republic of KoreaInstitute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju, Republic of KoreaLaccases are enzymes capable of oxidizing phenolic compounds and are important tools in different industrial processes. Heterologous expression of laccases is of great interest in biotechnological applications but achieving high expression levels is challenging. Three different laccases have been identified in the chestnut blight fungus Cryphonectria parasitica, among which a tannic acid-inducible laccase (laccase3) was successfully expressed using Saccharomyces cerevisiae. To obtain high and stable expression of fungal laccases, we cloned the gene encoding an extracellular laccase (Laccase1) of C. parasitica into a yeast episomal vector, used the resulting vectors to transform S. cerevisiae, and optimized the culture conditions of the selected transformants for Laccase1 production. We also tested the significance of the signal peptide of Laccase1 in the secretion of expressed Laccase1 and compared it with the widely used rice amylase signal peptide. Among the four constructs tested using a yeast episomal vector, full-length Laccase1 containing an endogenous signal peptide, showed the highest laccase activity. Interestingly, the stability of the recombinant vector expressing laccase was lower than that of the mock transformant, suggesting a detrimental effect of the Laccase1-expressing vector on host cells. Thus, we optimized the culture conditions to produce Laccase1 and the resulting optimum culture conditions identified through one-factor-at-a -time (OFAT) were 2% sucrose; 3% yeast nitrogen base without amino acid; pH 5.0; and 30 °C. The laccase activity was found to be 2.2 U/mL in optimal culture conditions, resulting in a 6.5-fold increase compared to the conventional culture medium.https://www.tandfonline.com/doi/10.1080/12298093.2024.2439646Laccase1Cryphonectria parasiticaSaccharomyces cerevisiaesignal peptideone-factor-at-a-time |
| spellingShingle | Kum-Kang So Fatima Alejandra Hernandez Alvarado Gui-Hwan Han Jeong-Won Kim Tae-Geum Kim Dae-Hyuk Kim Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae Mycobiology Laccase1 Cryphonectria parasitica Saccharomyces cerevisiae signal peptide one-factor-at-a-time |
| title | Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae |
| title_full | Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae |
| title_fullStr | Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae |
| title_full_unstemmed | Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae |
| title_short | Heterologous Expression of Laccase1 from Cryphonectria parasitica in Saccharomyces cerevisiae |
| title_sort | heterologous expression of laccase1 from cryphonectria parasitica in saccharomyces cerevisiae |
| topic | Laccase1 Cryphonectria parasitica Saccharomyces cerevisiae signal peptide one-factor-at-a-time |
| url | https://www.tandfonline.com/doi/10.1080/12298093.2024.2439646 |
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