Novel Transcription Activation Domains for Efficient Heterologous Gene Expression in Fungi

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http://urn.fi/URN:NBN:fi:hulib-202007033588
Title: Novel Transcription Activation Domains for Efficient Heterologous Gene Expression in Fungi
Author: Salumäe, Astrid
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2020
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202007033588
http://hdl.handle.net/10138/317447
Thesis level: master's thesis
Degree program: Genetiikan ja molekulaaristen biotieteiden maisteriohjelma
Master's Programme in Genetics and Molecular Biosciences
Magisterprogrammet i genetik och molekylära biovetenskaper
Specialisation: Biokemia ja rakennebiologia
Biochemistry and Structural biology
Biokemi och strukturbiologi
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.
Subject: Trichoderma reesei
Pichia pastoris
Zygosaccharomyces
synthetic transcription factor
expression
activation domain
CRISPR-Cas9
Cas9 D10A nickase
xylanase
phytase
protein production
secretion signal sequence
promoter


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