Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i>
Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. <i>Saccharomyces cerevisiae</i> is a preferred host for engineering fungal laccases. To assist the difficult secretion...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-01-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/22/3/1157 |
| _version_ | 1797407763095093248 |
|---|---|
| author | Pablo Aza Felipe de Salas Gonzalo Molpeceres David Rodríguez-Escribano Iñigo de la Fuente Susana Camarero |
| author_facet | Pablo Aza Felipe de Salas Gonzalo Molpeceres David Rodríguez-Escribano Iñigo de la Fuente Susana Camarero |
| author_sort | Pablo Aza |
| collection | DOAJ |
| description | Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. <i>Saccharomyces cerevisiae</i> is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of <i>S. cerevisiae</i> alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in <i>S. cerevisiae</i> of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of <i>S. cerevisiae</i> cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production. |
| first_indexed | 2024-03-09T03:47:19Z |
| format | Article |
| id | doaj.art-50065c23c4de4847a771e6cd53f1867c |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T03:47:19Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-50065c23c4de4847a771e6cd53f1867c2023-12-03T14:32:43ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-01223115710.3390/ijms22031157Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i>Pablo Aza0Felipe de Salas1Gonzalo Molpeceres2David Rodríguez-Escribano3Iñigo de la Fuente4Susana Camarero5Microbial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainMicrobial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainMicrobial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainMicrobial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainMicrobial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainMicrobial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, SpainLaccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. <i>Saccharomyces cerevisiae</i> is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of <i>S. cerevisiae</i> alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in <i>S. cerevisiae</i> of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of <i>S. cerevisiae</i> cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.https://www.mdpi.com/1422-0067/22/3/1157laccaseheterologous production<i>S. cerevisiae</i>directed evolutionsignal peptide<i>N</i>-glycosylation |
| spellingShingle | Pablo Aza Felipe de Salas Gonzalo Molpeceres David Rodríguez-Escribano Iñigo de la Fuente Susana Camarero Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> International Journal of Molecular Sciences laccase heterologous production <i>S. cerevisiae</i> directed evolution signal peptide <i>N</i>-glycosylation |
| title | Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> |
| title_full | Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> |
| title_fullStr | Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> |
| title_full_unstemmed | Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> |
| title_short | Protein Engineering Approaches to Enhance Fungal Laccase Production in <i>S. cerevisiae</i> |
| title_sort | protein engineering approaches to enhance fungal laccase production in i s cerevisiae i |
| topic | laccase heterologous production <i>S. cerevisiae</i> directed evolution signal peptide <i>N</i>-glycosylation |
| url | https://www.mdpi.com/1422-0067/22/3/1157 |
| work_keys_str_mv | AT pabloaza proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei AT felipedesalas proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei AT gonzalomolpeceres proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei AT davidrodriguezescribano proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei AT inigodelafuente proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei AT susanacamarero proteinengineeringapproachestoenhancefungallaccaseproductioniniscerevisiaei |