Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i>
Converting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain <i>Saccharomyces cerevisi...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | Journal of Fungi |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2309-608X/8/5/418 |
| _version_ | 1797498763240013824 |
|---|---|
| author | Anita Boisramé Cécile Neuvéglise |
| author_facet | Anita Boisramé Cécile Neuvéglise |
| author_sort | Anita Boisramé |
| collection | DOAJ |
| description | Converting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain <i>Saccharomyces cerevisiae</i> strains capable of metabolising pentose sugars mainly found in hemicellulose, enzymatic hydrolysis after pretreatment is still required. <i>Blastobotrys raffinosifermentans</i>, which naturally assimilates xylose and arabinose and displays numerous glycoside hydrolases, is a good candidate for direct and efficient conversion of renewable biomass. However, a greater diversity of tools for genetic engineering is needed. Here, we report the characterisation of four new promising promoters, a new dominant marker, and two vectors for the secretion of epitope tagged proteins along with a straightforward transformation protocol. The <i>TDH3</i> promoter is a constitutive promoter stronger than <i>TEF1</i>, and whose activity is maintained at high temperature or in the presence of ethanol. The regulated promoters respond to high temperature for <i>HSP26</i>, gluconeogenic sources for <i>PCK1</i> or presence of xylose oligomers for <i>XYL1</i>. Two expression/secretion vectors were designed based on p<i>TEF1</i> and p<i>TDH3</i>, two endogenous signal peptides from an α-arabinanase and an α-glucuronidase, and two epitopes. A heterologous α-arabinoxylan hydrolase from <i>Apiotrichum siamense</i> was efficiently secreted using these two vectors. |
| first_indexed | 2024-03-10T03:38:01Z |
| format | Article |
| id | doaj.art-ce794126b2274b67af860f9672a272f3 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T03:38:01Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-ce794126b2274b67af860f9672a272f32023-11-23T11:40:45ZengMDPI AGJournal of Fungi2309-608X2022-04-018541810.3390/jof8050418Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i>Anita Boisramé0Cécile Neuvéglise1SPO, INRAE, Institut Agro, Univ Montpellier, 34060 Montpellier, FranceSPO, INRAE, Institut Agro, Univ Montpellier, 34060 Montpellier, FranceConverting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain <i>Saccharomyces cerevisiae</i> strains capable of metabolising pentose sugars mainly found in hemicellulose, enzymatic hydrolysis after pretreatment is still required. <i>Blastobotrys raffinosifermentans</i>, which naturally assimilates xylose and arabinose and displays numerous glycoside hydrolases, is a good candidate for direct and efficient conversion of renewable biomass. However, a greater diversity of tools for genetic engineering is needed. Here, we report the characterisation of four new promising promoters, a new dominant marker, and two vectors for the secretion of epitope tagged proteins along with a straightforward transformation protocol. The <i>TDH3</i> promoter is a constitutive promoter stronger than <i>TEF1</i>, and whose activity is maintained at high temperature or in the presence of ethanol. The regulated promoters respond to high temperature for <i>HSP26</i>, gluconeogenic sources for <i>PCK1</i> or presence of xylose oligomers for <i>XYL1</i>. Two expression/secretion vectors were designed based on p<i>TEF1</i> and p<i>TDH3</i>, two endogenous signal peptides from an α-arabinanase and an α-glucuronidase, and two epitopes. A heterologous α-arabinoxylan hydrolase from <i>Apiotrichum siamense</i> was efficiently secreted using these two vectors.https://www.mdpi.com/2309-608X/8/5/418promoterxylanCAzymeyeast<i>Blastobotrys</i> <i>yvelinesensis</i> nomen nudumcell factory |
| spellingShingle | Anita Boisramé Cécile Neuvéglise Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> Journal of Fungi promoter xylan CAzyme yeast <i>Blastobotrys</i> <i>yvelinesensis</i> nomen nudum cell factory |
| title | Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> |
| title_full | Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> |
| title_fullStr | Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> |
| title_full_unstemmed | Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> |
| title_short | Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus <i>Blastobotrys</i> |
| title_sort | development of a vector set for high or inducible gene expression and protein secretion in the yeast genus i blastobotrys i |
| topic | promoter xylan CAzyme yeast <i>Blastobotrys</i> <i>yvelinesensis</i> nomen nudum cell factory |
| url | https://www.mdpi.com/2309-608X/8/5/418 |
| work_keys_str_mv | AT anitaboisrame developmentofavectorsetforhighorinduciblegeneexpressionandproteinsecretionintheyeastgenusiblastobotrysi AT cecileneuveglise developmentofavectorsetforhighorinduciblegeneexpressionandproteinsecretionintheyeastgenusiblastobotrysi |