ArticleMicrobial cell factories2019
CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.
Article in Microbial cell factories, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 65 papers.
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Who cites it
65 citing papers in PubMed.
- Development and characterization of a Cre/Synthetic and systems biotechnology · 2027Article
- Yeast species resource exploration and application in cell factories.Synthetic and systems biotechnology · 2026Review
- An advanced genome engineering platform for Komagataella phaffii enabling versatile in vivo DNA assembly and multiplex integration.Journal of biological engineering · 2026Article
- Harnessing the PpHog1 Regulatory Network to Engineer a UPR-Adaptive Komagataella phaffii Chassis for High-Copy Secretory Protein Production.Microbial biotechnology · 2026Article
- rDNA-mediated multicopy integration and gene dosage quantification system for microbial zeaxanthin biosynthesis.World journal of microbiology & biotechnology · 2026Article
- Systematic metabolic engineering of Komagataella phaffii for methanol-to-lactate bioconversion.Bioprocess and biosystems engineering · 2026Article
- Characterizing the role of an endogenous serine protease KpSub2 in recombinant collagen degradation in Komagataella phaffii.Bioresources and bioprocessing · 2026Article
- Domestication of Tartary Buckwheat Shaped a Regulatory Module for Seedling Salt Tolerance by Targeting the Magnesium Transporter Gene FtMGT2.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Engineering energy-efficient Saccharomyces cerevisiae for methanol and CONature communications · 2026Article
- CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies inSynthetic and systems biotechnology · 2025Article
- A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.Microbial cell factories · 2025Article
- Article
- SHIP identifies genomic safe harbors in eukaryotic organisms using genomic general feature annotation.Scientific reports · 2025Article
- Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid (3-HP) production from methanol.Journal of biological engineering · 2025Article
- Advancing recombinant protein expression in Komagataella phaffii: opportunities and challenges.FEMS yeast research · 2025Review
- Metabolic Engineering ofMolecules (Basel, Switzerland) · 2024Article
- Comparative omics directed gene discovery and rewiring for normal temperature-adaptive red pigment synthesis by polar psychrotrophic fungusSynthetic and systems biotechnology · 2024Article
- De novo biosynthesis and nicotinamide biotransformation of nicotinamide mononucleotide by engineered yeast cells.Microbial biotechnology · 2024Article
- Metabolic and tolerance engineering of Komagataella phaffii for 2-phenylethanol production through genome-wide scanning.Biotechnology for biofuels and bioproducts · 2024Article
- Applications of the Methylotrophic YeastJournal of fungi (Basel, Switzerland) · 2024Review
5 more citing papers are in PubMed but not listed here.
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8 authors.
Funding
Abstract
backgroundPichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.
resultsA non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (P
conclusionsA CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.
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