Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei.
ATP phosphoribosyltransferase
Gene targeting
Heterologous expression
Histidine auxotrophy
Marker recycling
Trichoderma reesei
Journal
Fungal biology and biotechnology
ISSN: 2054-3085
Titre abrégé: Fungal Biol Biotechnol
Pays: England
ID NLM: 101655873
Informations de publication
Date de publication:
14 Sep 2021
14 Sep 2021
Historique:
received:
06
07
2021
accepted:
06
09
2021
entrez:
15
9
2021
pubmed:
16
9
2021
medline:
16
9
2021
Statut:
epublish
Résumé
The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei. In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci. With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.
Sections du résumé
BACKGROUND
BACKGROUND
The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei.
RESULTS
RESULTS
In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci.
CONCLUSION
CONCLUSIONS
With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.
Identifiants
pubmed: 34521467
doi: 10.1186/s40694-021-00116-5
pii: 10.1186/s40694-021-00116-5
pmc: PMC8442374
doi:
Types de publication
Journal Article
Langues
eng
Pagination
9Subventions
Organisme : austrian science fund
ID : P 34036
Informations de copyright
© 2021. The Author(s).
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