ArticlePloS one2012
Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 127 papers.
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127 citing papers in PubMed, 297 citations in OpenAlex.
- Yarrowia lipolytica and Komagataella phaffii (Pichia pastoris) as Hosts for Heterologous Expression of Lipases.Methods in molecular biology (Clifton, N.J.) · 2027Article
- Metabolic engineering ofApplied and environmental microbiology · 2026Review
- An advanced genome engineering platform for Komagataella phaffii enabling versatile in vivo DNA assembly and multiplex integration.Journal of biological engineering · 2026Article
- Enhanced secretion of thermostable phytases from Myceliophthora thermophila by Komagataella phaffii.Microbial cell factories · 2026Article
- Multistep metabolic engineering of Pichia pastoris for biosynthesis of N‑acetylneuraminic acid.Applied microbiology and biotechnology · 2026Article
- Enzymatic and environmental degradation of natural rubber-PHBV polymer blends.Biodegradation · 2026Article
- Engineering small peptide secretion inFrontiers in bioengineering and biotechnology · 2026Article
- Classical Genetics of Pichia pastoris.Methods in molecular biology (Clifton, N.J.) · 2026Article
- In Vivo Plasmid Assembly in NHEJ-Deficient Pichia pastoris Strains.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Cloning of MFα Secretion Signal Variants for Protein Secretion by Pichia pastoris.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Auxin-Induced Degradation of Target Proteins in Pichia pastoris.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Pichia pastoris Genomic Resources: Accessibility, Recommendations, and Pitfalls.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Toward food-grade production of the Glutamicibacter halophytocola diamine oxidase using Komagataella phaffii.AMB Express · 2025Article
- Next-generation stress-inducible Komagataella phaffii promoter variants.Microbial cell factories · 2025Article
- Sterol regulation in Komagataella phaffii: Identification of sterol regulatory element binding proteins and their activation pathway.The Journal of biological chemistry · 2025Article
- Utilizing native nanodiscs to isolate active TRPC3 channels and expand structural analysis capabilities.Scientific reports · 2025Article
- The MFα signal sequence in yeast-based protein secretion: challenges and innovations'.Applied microbiology and biotechnology · 2025Review
- A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.Microbial cell factories · 2025Article
- Considerations for Domestication of Novel Strains of Filamentous Fungi.ACS synthetic biology · 2025Review
- Advances in CRISPR-enabled genome-wide screens in yeast.FEMS yeast research · 2025Review
67 more citing papers are in PubMed but not listed here.
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Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts.
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