Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii

Lehmayer, Leonie; Bernauer, Lukas; Emmerstorfer-Augustin, Anita

doi:10.1016/j.isci.2022.104888

Cited by 3 publications

(2 citation statements)

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“…Bands of the correct size were gel-purified. The transformation of K. phaffii was performed according to the condensed protocol of Lin-Cereghino et al [62]. For gene editing using CRISPR/Cas9, we followed the protocol of Weninger at al., 2016 [63].…”

Section: Cloning and Strain Constructionmentioning

confidence: 99%

Human Sterols Are Overproduced, Stored and Excreted in Yeasts

Radkohl,

Schusterbauer,

Bernauer

et al. 2024

IJMS

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Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in K. phaffii, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.

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Section: Cloning and Strain Constructionmentioning

confidence: 99%

Human Sterols Are Overproduced, Stored and Excreted in Yeasts

Radkohl,

Schusterbauer,

Bernauer

et al. 2024

IJMS

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“…As explained above, a rich portfolio of interesting enzymes can already be produced using K. phaffii at industrial scale by applying a diverse range of engineering approaches, depending on the protein to be produced. In the future, the possibilities of strain engineering will become even more prominent thanks to advanced strain engineering strategies, including CRISPR/Cas9 [ 159 ], CRISPRi [ 160 ], or the auxin-inducible degron (AID)-technology [ 161 ]. Although, CRISPR/Cas9 and related technologies allow for efficient and targeted strain engineering, CRISPRi can be used to either repress or induce a target gene.…”

Section: Emerging Trends Of the Biotechnological Applications Via ...mentioning

confidence: 99%

Industrial Production of Proteins with Pichia pastoris—Komagataella phaffii

Barone

Emmerstorfer-Augustin

Biundo

et al. 2023

Biomolecules

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Since the mid-1960s, methylotrophic yeast Komagataella phaffii (previously described as Pichia pastoris) has received increasing scientific attention. The interest for the industrial production of proteins for different applications (e.g., feed, food additives, detergent, waste treatment processes, and textile) is a well-consolidated scientific topic, and the importance for this approach is rising in the current era of environmental transition in human societies. This review aims to summarize fundamental and specific information in this scientific field. Additionally, an updated description of the relevant products produced with K. phaffii at industrial levels by a variety of companies—describing how the industry has leveraged its key features, from products for the ingredients of meat-free burgers (e.g., IMPOSSIBLE™ FOODS, USA) to diabetes therapeutics (e.g., Biocon, India)—is provided. Furthermore, active patents and the typical workflow for industrial protein production with this strain are reported.

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