Browsing by Subject "protein production"

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  • Claassens, Nico J.; Finger-Bou, Max; Scholten, Bart; Muis, Frederieke; de Groot, Jonas J.; de Gier, Jan-Willem; de Vos, Willem M.; van der Oost, John (2019)
    Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.
  • Salumäe, Astrid (Helsingin yliopisto, 2020)
    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.