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Utilizing Komagataella phaffii (K. phaffii) as a host, methanol-dependent fed-batch cultivations remain state-of-the-art for recombinant protein production. Recently, however, derepressible promoters have emerged as a valuable methanol-free alternative, especially for the expression of complex target proteins. In this study, we investigated the expression of a recombinant model enzyme (UPO) using a derepressible bi-directionalized promoter system in continuous cultivations. According to the literature, low growth rates required for derepression might result in pseudohyphae growth in chemostat cultivations with K. phaffii. This phenotype would be highly undesired as pseudohyphae growth is referred to decreasing productivity. Still, literature on derepressible promoter systems used in continuous cultivations is scarce. Hence, we aim to investigate pseudohyphae growth in a derepressible bi-directionalized promoter system. Several chemostats and a decelerostat screening were performed to identify the effect of the specific growth rate on pseudohyphae growth in continuous cultivations whilst monitoring the productivity of the recombinant target enzyme. Based on the experimental screening data, derepression was still achieved at a growth rate of 0.11 h-1, whilst no pseudohyphae growth was observed. However, verifying these conditions for an extended timeframe of more than five residence times triggered pseudohyphae formation. Hence, the results of this study indicate that pseudohyphae growth in chemostats with derepressible promoter systems in K. phaffii is both growth-rate and time-dependent, thus limiting the potential of continuous cultivations for recombinant production of UPO. Despite the observed limitations, we still propose decelerostat cultivations as a proper screening tool to determine suitable production conditions in continuous systems for derepressed promotors. more...

Digital media have become an integral part of everyday life, education and work. However, intensive and problematic media use, and in particular problematic smartphone use has been shown to reliably predict reduced well-being and increased stress. Therefore, it is essential to investigate the factors that lead to problematic smartphone use and respective negative well-being outcomes and to develop interventions that effectively address these factors. Considering self-control and fear of missing out (FoMO) as key psychological factors promoting problematic smartphone use, we present a conceptual model explaining the emergence of digital stress due to problematic smartphone use, upon which we built a blended training intervention against digital stress. A controlled trial evaluation of the training intervention provided data at multiple time points for multilevel regression analyses on a sample of 175 university students. The results indicated that the intervention was effective in reducing FoMO (fear of missing out) and increasing self-control. Indirect effects suggested that both the reduction in FoMO and the gain in self-control effectively reduced emotional irritation and perceived stress via a reduction in problematic smartphone use. Conclusively, the findings identify key psychological factors that promote problematic smartphone use and demonstrate that these factors can be effectively addressed through appropriate psychological interventions. more...

Abstract Background Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. Results In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. Conclusions In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis. more...

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (PAOX1) and/or the constitutive GAP promoter (PGAP). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical PGAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed PGAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the PGAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than PGAP fed-batch at low μ. Compared to the optimum approach for PGAP-based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy. more...

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods. more...

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Die überfachlichen Kompetenzen von Studierenden zu fördern ist eine Kernaufgabe von Universitäten. Die Bologna-Reformen haben zu tief greifenden Veränderungen des studentischen Lebens und Arbeitens geführt, und intelligente Maßnahmen zur Kompetenzförderung werden immer notwendiger, um Studienabbrüchen entgegenzuwirken und die erfolgreiche Beendigung des Studiums zu unterstützen. Die Implementierung von Coaching für Studenten unterstützt die nachhaltige Förderung von Selbstmanagementkompetenzen an Universitäten unabhängig von Fachrichtung oder Vorbildung. Der folgende Beitrag erörtert nicht nur die grundlegenden Erfolgsfaktoren für studentisches Coaching, sondern weitet den Blick für einen innovativen Ansatz: Den Einsatz von Studenten als Coach. 10.03.2010 | Katharina Ebner (München) more...