Condensed Protocol for Competent Cell Preparation and Transformation of the Methylotrophic Yeast Pichia Pastoris

BioTechniquesVol. 38, No. 1 BenchmarksOpen AccessCondensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastorisJoan Lin-Cereghino, William W. Wong, See Xiong, William Giang, Linda T. Luong, Jane Vu, Sabrina D. Johnson & Geoff P. Lin-CereghinoJoan Lin-Cereghino*Address correspondence to: Joan Lin-Cereghino, Department of Biological Sciences, University of the Pacific, 1050 W. Brookside Road, Stockton, CA 95211, USA. e-mail: E-mail Address: jlincere@pacific.eduUniversity of the Pacific, Stockton, CA, USA, William W. WongUniversity of the Pacific, Stockton, CA, USA, See XiongUniversity of the Pacific, Stockton, CA, USA, William GiangUniversity of the Pacific, Stockton, CA, USA, Linda T. LuongUniversity of the Pacific, Stockton, CA, USA, Jane VuUniversity of the Pacific, Stockton, CA, USA, Sabrina D. JohnsonUniversity of the Pacific, Stockton, CA, USA & Geoff P. Lin-CereghinoUniversity of the Pacific, Stockton, CA, USAPublished Online:30 May 2018https://doi.org/10.2144/05381BM04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris—spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection.The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heat-shock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably.We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this "hybrid" method are then electroporated using the same parameters as conventional electroporation.Transformation efficiencies using the condensed protocol are comparable to the conventional electroporation procedure using auxotrophic markers but are approximately 20-fold lower using the zeocin resistance marker. However, the condensed protocol provides sufficient transformants, including multicopy integrants, for protein expression studies and has several advantages over the conventional electroporation and heat-shock methods. Table 1 compares the steps in cell preparation and transformation for conventional electroporation, heat shock, and our condensed protocol. Compared to the heat-shock method, the condensed protocol requires less time for the transformation step and provides much higher transformation efficiencies. Compared to the electroporation procedure, the new procedure saves both reagents and time during cell preparation. In addition, the fewer number of steps during the cell preparation of the condensed protocol reduce the chance of contamination of competent yeast cells. Furthermore, unlike the electroporation cell preparation procedure, our condensed protocol does not require a large, refrigerated centrifuge. We use a small, nonrefrigerated centrifuge capable of spinning six 50-mL conical tubes at a time, enabling us to quickly prepare competent cells of six different strains simultaneously. This is significant because some P. pastoris strains (i.e., protease deficient, methanol utilization deficient) can express a given protein more efficiently than others, and it is often necessary to transform the same expression plasmid into various strains to determine empirically which strain gives the highest expression. Thus, the condensed protocol (Table 2) enables a researcher to prepare and transform multiple samples of highly competent P. pastoris cells in a short time with minimal equipment or effort.Table 1. Comparison of Transformation ProtocolsTable 2. Condensed Protocol: Preparation of Competent Cells and TransformationUsing HIS4-based and zeocin-based plasmids based on typical P. pastoris cloning vectors, pHILA1 and pPICZB (Invitrogen, Carlsbad, CA, USA), and strains such as JC100 (wild-type) or GS115 (his4), we obtained the results summarized in Table 3. These results are average transformation efficiencies calculated from at least five separate transformations.Table 3. Transformation EfficienciesThe condensed protocol utilizes the most efficient portions of the electroporation and heat-shock transformation protocols to yield a procedure for P. pastoris cells that produces high transformation efficiencies while saving time, effort, and reagents.AcknowledgmentsThe authors would like to thank all members of the Lin-Cereghino laboratory for their support. This work was funded by undergraduate research funds from the University of the Pacific and National Institutes of Health (NIH)-AREA grant no. GM65882 to J.L.-C. and G.P.L.-C.Competing Interests StatementThe authors declare no competing interests.References1. Lin Cereghino, J. and J.M. Cregg. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24:45–66.Crossref, Medline, Google Scholar2. Cregg, J.M., K.J. Barringer, A.Y. Hessler, and K.R. Madden. 1985. Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5:3376–3385.Crossref, Medline, CAS, Google Scholar3. Cregg, J.M. and K.A. Russell. 1998. Transformation methods, p. 27–39. In D.R. Higgins and J.M. Cregg (Eds.), Methods in Molecular Methods Molecular Biology, Pichia Protocols, Vol. 103. Humana Press, Totowa, NJ.Google Scholar4. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163–168.Crossref, Medline, CAS, Google Scholar5. Dohmen, R.J., A.W.M. Strasser, C.B. Höner, and C.P. Hollenberg. 1991. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByCloning and characterization of nitrate reductase gene in kelp Saccharina japonica (Laminariales, Phaeophyta)6 February 2023 | BMC Plant Biology, Vol. 23, No. 1Transcriptomic profiling of the yeast Komagataella phaffii in response to environmental alkalinization4 April 2023 | Microbial Cell Factories, Vol. 22, No. 1Heterologous expression, purification, and characterization of a recombinant Cordyceps militaris lipase from Candida rugosa-like family in Pichia pastorisEnzyme and Microbial Technology, Vol. 168Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris19 November 2022 | Biotechnology Letters, Vol. 45, No. 1Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins15 December 2022 | PLOS Biology, Vol. 20, No. 12A switchable secrete-and-capture system enables efficient selection of Pichia pastoris clones producing high yields of Fab fragmentsJournal of Immunological Methods, Vol. 511Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden2 July 2022 | Scientific Reports, Vol. 12, No. 1Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris7 September 2022 | Microbial Cell Factories, Vol. 21, No. 1Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris14 May 2022 | Biotechnology for Biofuels and Bioproducts, Vol. 15, No. 1Challenges of expressing recombinant human tissue factor as a secreted protein in Pichia pastoris8 February 2022 | Preparative Biochemistry & Biotechnology, Vol. 52, No. 9Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress21 September 2022 | Journal of Fungi, Vol. 8, No. 10Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffiiiScience, Vol. 25, No. 9Auto-induction Screening Protocol for Ranking Clonal Libraries of Pichia pastoris MutS Strains20 July 2022 | Biotechnology and Bioprocess Engineering, Vol. 27, No. 4Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)27 July 2022 | Applied Sciences, Vol. 12, No. 15Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene22 June 2022 | Journal of Agricultural and Food Chemistry, Vol. 70, No. 26Racemization-free and scalable amidation of l -proline in organic media using ammonia and a biocatalyst only1 January 2022 | Green Chemistry, Vol. 24, No. 13Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism20 July 2022 | Microorganisms, Vol. 10, No. 7Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation SpectroscopyInternational Journal of Pharmaceutics, Vol. 621Bioengineering a glucose oxidase nanosensor for near-infrared continuous glucose monitoring1 January 2022 | Nanoscale Advances, Vol. 4, No. 11Synthesis of bioengineered heparin by recombinant yeast Pichia pastoris1 January 2022 | Green Chemistry, Vol. 24, No. 8High Production of Trametes cinnabarina Laccase (lac1) by Suspended and Immobilized Cells of Recombinant Pichia pastoris from Crude Glycerol26 January 2022 | Waste and Biomass Valorization, Vol. 13, No. 4Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase15 March 2022 | International Journal of Molecular Sciences, Vol. 23, No. 6Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms21 January 2022 | ACS Synthetic Biology, Vol. 11, No. 2Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas926 January 2022 | ACS Synthetic Biology, Vol. 11, No. 2Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity25 January 2022 | Molecules, Vol. 27, No. 3Competent Cell Preparation and Transformation of Pichia pastoris4 July 2022CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome4 July 2022Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris4 July 2022A highly versatile fungal glucosyltransferase for specific production of quercetin-7-O-β-d-glucoside and quercetin-3-O-β-d-glucoside in different hosts7 December 2021 | Applied Microbiology and Biotechnology, Vol. 106, No. 1Size‐Controlled Synthesis of β(1→4)‐GlcNAc Oligosaccharides Using an Endo‐Glycosynthase29 October 2021 | Chemistry – A European Journal, Vol. 27, No. 70A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris13 November 2021 | Applied Microbiology and Biotechnology, Vol. 105, No. 24A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases12 May 2021 | Communications Biology, Vol. 4, No. 1Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris23 March 2021 | Microbial Cell Factories, Vol. 20, No. 1Two Fusarium copper radical oxidases with high activity on aryl alcohols16 June 2021 | Biotechnology for Biofuels, Vol. 14, No. 1Modular biosynthesis of plant hemicellulose and its impact on yeast cells19 June 2021 | Biotechnology for Biofuels, Vol. 14, No. 1Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain29 September 2021 | Antibodies, Vol. 10, No. 4Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccineVaccine, Vol. 39, No. 51Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris7 January 2021 | Microbial Cell Factories, Vol. 20, No. 1Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris31 March 2021 | Biotechnology for Biofuels, Vol. 14, No. 1Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C926 April 2021 | Microbial Cell Factories, Vol. 20, No. 1Improved Production of Recombinant Myrosinase in Pichia pastoris2 November 2021 | International Journal of Molecular Sciences, Vol. 22, No. 21Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion MutationsApplied and Environmental Microbiology, Vol. 87, No. 19Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris1 July 2021 | Nucleic Acids Research, Vol. 49, No. 13Alternative Routes for the Production of Natural 4-Vinylguaiacol from Sugar Beet Fiber Using Basidiomycetous Enzymes14 May 2021 | Catalysts, Vol. 11, No. 5Optimisation of Recombinant Myrosinase Production in Pichia pastoris1 April 2021 | International Journal of Molecular Sciences, Vol. 22, No. 7A Potential Role for Fructosamine-3-Kinase in Cataract Treatment7 April 2021 | International Journal of Molecular Sciences, Vol. 22, No. 8Knockout of RSN1 , TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris12 December 2020 | Traffic, Vol. 22, No. 3Molecular cloningThe TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and ArabidopsisPlant Science, Vol. 302Expression of proteins in Pichia pastoris'Applying the Auxin-Based Degron System for the Inducible, Reversible and Complete Protein Degradation in Komagataella phaffii'SSRN Electronic JournalGeneration of 4-vinylguaiacol through a novel high-affinity ferulic acid decarboxylase to obtain smoke flavours without carcinogenic contaminants21 December 2020 | PLOS ONE, Vol. 15, No. 12Enhancement of cell proliferation and motility of mammalian cells grown in co-culture with Pichia pastoris expressing recombinant human FGF-2Protein Expression and Purification, Vol. 176Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale4 May 2020 | Scientific Reports, Vol. 10, No. 1Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris25 February 2020 | AMB Express, Vol. 10, No. 1Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B3 February 2020 | Scientific Reports, Vol. 10, No. 1Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing25 November 2020 | Frontiers in Bioengineering and Biotechnology, Vol. 8Secretion of Pseudomonas aeruginosa Lipoxygenase by Pichia pastoris upon Glycerol Feed7 October 2020 | Biotechnology Journal, Vol. 15, No. 11Modified chemical method for efficient transformation and diagnosis in Pichia pastorisProtein Expression and Purification, Vol. 174Synergistic codon optimization and bioreactor cultivation toward enhanced secretion of fungal lignin peroxidase in Pichia pastoris: Enzymatic valorization of technical (industrial) ligninsEnzyme and Microbial Technology, Vol. 139Immobilization of unspecific peroxygenase expressed in Pichia pastoris by metal affinity bindingMolecular Catalysis, Vol. 492Real-time monitoring of population dynamics and physical interactions in a synthetic yeast ecosystem by use of multicolour flow cytometry21 April 2020 | Applied Microbiology and Biotechnology, Vol. 104, No. 12Engineering and optimization of phosphate-responsive phytase expression in Pichia pastoris yeast for phytate hydrolysisEnzyme and Microbial Technology, Vol. 137Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid AntibodiesCell, Vol. 181, No. 5A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity27 March 2020 | Molecules, Vol. 25, No. 7Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain12 November 2019 | Biotechnology and Bioengineering, Vol. 117, No. 2Host-Informed Expression of CRISPR Guide RNA for Genomic Engineering in Komagataella phaffii11 December 2019 | ACS Synthetic Biology, Vol. 9, No. 1Structure/Function Analysis of human ZnT8 (SLC30A8): A Diabetes Risk Factor and Zinc TransporterCurrent Research in Structural Biology, Vol. 2Thermostability improvement of Aspergillus awamori glucoamylase via directed evolution of its gene located on episomal expression vector in Pichia pastoris cells31 December 2019 | Protein Engineering, Design and Selection, Vol. 32, No. 6Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection12 December 2019 | Frontiers in Immunology, Vol. 10Role of BGS13 in the Secretory Mechanism of Pichia pastorisApplied and Environmental Microbiology, Vol. 85, No. 24Engineered Pichia pastoris production of fusaruside, a selective immunomodulator17 June 2019 | BMC Biotechnology, Vol. 19, No. 1Heterologous coexpression of the benzoate‐para‐hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts19 October 2018 | Microbial Biotechnology, Vol. 12, No. 6Production of a Recombinant Non-Hydroxylated Gelatin Mimetic in Pichia pastoris for Biomedical Applications2 September 2019 | Journal of Functional Biomaterials, Vol. 10, No. 3Pichia pastoris protease‐deficient and auxotrophic strains generated by a novel, user‐friendly vector toolbox for gene deletion30 July 2019 | Yeast, Vol. 98Pichia pastoris as a host for production and isolation of mutagenic AID/APOBEC enzymes involved in cancer and immunityNew Biotechnology, Vol. 51Simplified protocol for faster transformation of (a large number of) Pichia pastoris strains13 March 2019 | Yeast, Vol. 36, No. 6Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris27 May 2019 | World Journal of Microbiology and Biotechnology, Vol. 35, No. 6Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris15 February 2019 | Yeast, Vol. 36, No. 5Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene‐expression differences between Mut+ and MutS strains of Pichia pastoris13 June 2019 | Yeast, Vol. 36, No. 5Novel α-1,3/α-1,4-Glucosidase from Aspergillus niger Exhibits Unique Transglucosylation to Generate High Levels of Nigerose and Kojibiose26 February 2019 | Journal of Agricultural and Food Chemistry, Vol. 67, No. 12Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity20 September 2018 | Biotechnology Journal, Vol. 14, No. 3Long-Term Monokaryotic Cultures of Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene11 August 2018 | Applied Biochemistry and Biotechnology, Vol. 187, No. 3Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris27 February 2019 | Frontiers in Microbiology, Vol. 10Membrane Protein Production in Yeast: Modification of Yeast Membranes for Human Membrane Protein Production9 February 2019High-Throughput Screening and Selection of Pichia pastoris Strains9 February 2019Non-Conventional Yeast Species for Recombinant Protein and Metabolite ProductionWhole-cell (+)-ambrein production in the yeast Pichia pastorisMetabolic Engineering Communications, Vol. 7Engineered bidirectional promoters enable rapid multi-gene co-expression optimization4 September 2018 | Nature Communications, Vol. 9, No. 1Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L.Journal of Biotechnology, Vol. 284Production of Ready-To-Use Functionalized Sup35 Nanofibrils Secreted by Komagataella pastoris12 September 2018 | ACS Nano, Vol. 12, No. 9BadGluc, a β-glucosidase from Bjerkandera adusta with anthocyanase properties12 June 2018 | Bioprocess and Biosystems Engineering, Vol. 41, No. 9Engineering of the upper hinge region of human IgG1 Fc enhances the binding affinity to FcγIIIa (CD16a) receptor isoform9 October 2018 | Protein Engineering, Design and Selection, Vol. 31, No. 6Optimization of Δ 9 -tetrahydrocannabinolic acid synthase production in Komagataella phaffii via post-translational bottleneck identificationJournal of Biotechnology, Vol. 272-273Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastorisApplied and Environmental Microbiology, Vol. 84, No. 6Pichia pastoris Alcohol Oxidase 1 ( AOX1 ) Core Promoter Engineering by High Resolution Systematic Mutagenesis22 November 2017 | Biotechnology Journal, Vol. 13, No. 3Production of Hydroxynitrile Lyase from Davallia tyermannii ( Dt HNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst11 December 2017 | ChemBioChem, Vol. 19, No. 4Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca8 February 2018 | PLOS ONE, Vol. 13, No. 2Construction of a cellulose-metabolizing Komagataella phaffii (Pichia pastoris) by co-expressing glucanases and β-glucosidase4 December 2017 | Applied Microbiology and Biotechnology, Vol. 102, No. 3Cell-free one-pot conversion of (+)-valencene to (+)-nootkatone by a unique dye-decolorizing peroxidase combined with a laccase from Funalia trogii1 February 2018 | Journal of Industrial Microbiology and Biotechnology, Vol. 45, No. 2Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris10 October 2017 | Biotechnology Letters, Vol. 40, No. 1The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide AssayBioMed Research International, Vol. 2018A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris17 November 2017 | Scientific Reports, Vol. 7, No. 1A high-throughput expression screening platform to optimize the production of antimicrobial peptides13 February 2017 | Microbial Cell Factories, Vol. 16, No. 1Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris31 July 2017 | Applied Microbiology and Biotechnology, Vol. 101, No. 18Variants of PpuLcc, a multi-dye decolorizing laccase from Pleurotus pulmonarius expressed in Pichia pastorisProtein Expression and Purification, Vol. 137Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe3 August 2017 | Bioscience, Biotechnology, and Biochemistry, Vol. 81, No. 8A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activityNew Biotechnology, Vol. 37A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors11 May 2017 | ACS Infectious Diseases, Vol. 3, No. 6A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity31 May 2017 | PLOS ONE, Vol. 12, No. 5Engineering microbial phenotypes through rewiring of genetic networks21 March 2017 | Nucleic Acids Research, Vol. 45, No. 8Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state13 February 2017 | Nature Communications, Vol. 8, No. 1Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris14 December 2016 | Applied Microbiology and Biotechnology, Vol. 101, No. 7Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species14 December 2016 | ACS Synthetic Biology, Vol. 6, No. 3Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastorisProtein Expression and Purification, Vol. 130Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris14 April 2016 | Biotechnology and Applied Biochemistry, Vol. 64, No. 1Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?Biologicals, Vol. 45Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastorisGene, Vol. 598Characterization of human S-adenosyl-homocysteine hydrolase in vitro and identification of its potential inhibitors21 September 2017 | Journal of Enzyme Inhibition and Medicinal Chemistry, Vol. 32, No. 1Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane proteinBiochemical and Biophysical Research Communications, Vol. 481, No. 1-2Pichia pastoris engineering for the production of a modified phospholipase CProcess Biochemistry, Vol. 51, No. 12Pichia pastoris MutS strains are prone to misincorporation of O-methyl-l-homoserine at methionine residues when methanol is used as the sole carbon source7 June 2016 | Microbial Cell Factories, Vol. 15, No. 1Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications11 August 2016 | Microbial Cell Factories, Vol. 15, No. 1Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters20 October 2015 | Biotechnology and Applied Biochemistry, Vol. 63, No. 6Feruloyl esterases from Schizophyllum commune to treat food industry side-streamsBioresource Technology, Vol. 220Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastorisProtein Expression and Purification, Vol. 127Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastorisJournal of Biotechnology, Vol. 235Pichia pastoris mutants as host strains for efficient secretion of recombinant branched chain aminotransferase (BCAT)Journal of Biotechnology, Vol. 235The PARS sequence increase the efficiency of stable Pichia pastoris transformationJournal of Microbiological Methods, Vol. 129Biotechnological advances towards an enhanced peroxidase production in Pichia pastorisJournal of Biotechnology, Vol. 233Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger1 September 2016 | Bioscience, Biotechnology, and Biochemistry, Vol. 80, No. 9Effect of chloride ions on the catalytic properties of human pancreatic α-amylase isozyme produced in Pichia pastorisKorean Journal of Food Science and Technology, Vol. 48, No. 4Centromeres of the Yeast Komagataella phaffii ( Pichia pastoris) Have a Simple Inverted-Repeat Structure6 August 2016 | Genome Biology and Evolution, Vol. 8, No. 8GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris4 May 2016 | Biotechnology Progress, Vol. 32, No. 4Heterologous expression of Aspergillus terreus fructosyltransferase in Kluyveromyces lactisNew Biotechnology, Vol. 33, No. 4Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics13 January 2016 | Applied Microbiology and Biotechnology, Vol. 100, No. 10Heterologous production of the stain solving peptidase PPP1 from