Novel Transcription Activation Domains for Efficient Heterologous Gene Expression in Fungi

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dc.contributor Helsingin yliopisto, Bio- ja ympäristötieteellinen tiedekunta fi
dc.contributor University of Helsinki, Faculty of Biological and Environmental Sciences en
dc.contributor Helsingfors universitet, Bio- och miljövetenskapliga fakulteten sv Salumäe, Astrid 2020
dc.identifier.uri URN:NBN:fi:hulib-202007033588
dc.description.abstract In biotechnological protein production and metabolic engineering, regulating the expression of genes is essential. For this, expression systems composed of promoters, terminators and transcription factors are essential. So far, majority of these systems use native promoters and transcription factors. That however rises two problems: 1) these systems usually work in only a set of closely related species, 2) native regulatory components can cause unintended expression levels due to the complexity of cellular regulation. Recently, a synthetic expression system (SES) was established for a wide range of fungal species. The transcription factor used in this system comprises an activation domain that originates from a virus. However, in the field of biotechnology and especially food industry, viral DNA constructs are not favorable because of customer concerns. In this paper, plant-derived activation domains were screened in Trichoderma reesei and Pichia pastoris using mCherry as a target gene for measuring the expression levels. The best expression systems were also tested for protein production in T. reesei and P. pastoris. We tested the production of two different proteins – a bacterial xylanase and a phytase. Two of the novel activation domains provided similar expression levels to the viral activation domain in both fungi. In addition, we developed optimized expression systems for an unconventional yeast from Zygosaccharomyces spp. using the novel transcription factors. The best SES version was used for secretion signal sequence screening for xylanase protein production. To further improve the use of T. reesei as a production host, the CRISPR-Cas9 system with the Cas9 D10A nickase version was tested for transformation of T. reesei. Here, we demonstrated the genomic integration and expression of Cas9 D10A nickase in T. reesei using the SES system with the novel plant-derived activation domain. Furthermore, we successfully transformed the T. reesei Cas9 D10A nickase expressing strain using only guide-RNAs and a donor DNA. en
dc.language.iso eng
dc.publisher Helsingin yliopisto fi
dc.publisher University of Helsinki en
dc.publisher Helsingfors universitet sv
dc.subject Trichoderma reesei en
dc.subject Pichia pastoris en
dc.subject Zygosaccharomyces en
dc.subject synthetic transcription factor en
dc.subject expression en
dc.subject activation domain en
dc.subject CRISPR-Cas9 en
dc.subject Cas9 D10A nickase en
dc.subject xylanase en
dc.subject phytase en
dc.subject protein production en
dc.subject secretion signal sequence en
dc.subject promoter en
dc.title Novel Transcription Activation Domains for Efficient Heterologous Gene Expression in Fungi en
dc.type.ontasot pro gradu -tutkielmat fi
dc.type.ontasot master's thesis en
dc.type.ontasot pro gradu-avhandlingar sv
dct.identifier.urn URN:NBN:fi:hulib-202007033588
dc.subject.specialization Biokemia ja rakennebiologia fi
dc.subject.specialization Biochemistry and Structural biology en
dc.subject.specialization Biokemi och strukturbiologi sv
dc.subject.degreeprogram Genetiikan ja molekulaaristen biotieteiden maisteriohjelma fi
dc.subject.degreeprogram Master's Programme in Genetics and Molecular Biosciences en
dc.subject.degreeprogram Magisterprogrammet i genetik och molekylära biovetenskaper sv

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