{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T06:04:33Z","timestamp":1789538673067,"version":"build-2803163510"},"reference-count":60,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T00:00:00Z","timestamp":1789516800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008332","name":"TU Graz, Internationale Beziehungen und Mobilit\u00e4tsprogramme","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008332","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Small peptides have emerged as an important class of biologics with broad therapeutic applications. Limitations of conventional solid-phase synthesis have intensified the interest in microbial production systems.\n                      <jats:italic>Komagataella phaffii<\/jats:italic>\n                      , a widely used host for secretory protein production, represents a promising alternative platform; however, the efficient secretion of very small peptides (&amp;lt;3.5\u00a0kDa) remains a major biotechnological challenge due to constraints in detection, proteolytic processing, and overall secretion efficiency.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>To systematically investigate bottlenecks in small peptide secretion, we established the 3\u00d7FLAG peptide (23 amino acids) as a tractable, immunologically detectable model system. We evaluated the impact of peptide chain length and signal sequence selection on secretory performance. Furthermore, we assessed the effect of overexpressing the translocon components and conducted a random mutagenesis screen to identify novel genetic determinants influencing secretion. Finally, we tested the transferability of our engineering strategies on the disulfide-rich peptide dQ-brazzein and the therapeutic fusion peptides \u03b1-melanocyte-stimulating hormone and salmon calcitonin.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Secretion efficiency exhibited a strong dependency on peptide chain length and signal sequence selection, likely reflecting limitations during endoplasmic reticulum translocation. Overexpression of Sec61 partially alleviated this bottleneck, enhancing secretion approximately 2-fold. Through the random mutagenesis screen, we identified\n                      <jats:italic>AQR1<\/jats:italic>\n                      ,\n                      <jats:italic>FMS1-2<\/jats:italic>\n                      ,\n                      <jats:italic>SKG3<\/jats:italic>\n                      , and\n                      <jats:italic>CDC48<\/jats:italic>\n                      as novel targets, which increased secretion up to 9-fold. These engineering strategies were tested on dQ-brazzein, yielding up to \u223c100% improvement, though the magnitude of transfer was cargo-dependent. Furthermore, the pronounced secretion limitations observed for \u03b1-melanocyte-stimulating hormone and salmon calcitonin based fusion peptides were partially mitigated utilizing these strategies, resulting in a \u223c2-fold improvement.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This study provides a systematic evaluation of engineering strategies targeting the unique constraints of very small (&amp;lt;3.5\u00a0kDa) peptide secretion in\n                      <jats:italic>K. phaffii<\/jats:italic>\n                      . The identified genetic targets and mechanistic insights serve as a starting point for cargo-specific optimization efforts and highlight the unique challenges that short peptides pose to the native secretory machinery.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fbioe.2026.1929744","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:30:42Z","timestamp":1789536642000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering small peptide secretion in Komagataella phaffii"],"prefix":"10.3389","volume":"14","author":[{"given":"Johanna","family":"Pirchner","sequence":"first","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon","family":"Arhar","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Corinna","family":"Odar","sequence":"additional","affiliation":[{"name":"Austrian Centre of Industrial Biotechnology, acib GmbH","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa","family":"Fr\u00fchauf","sequence":"additional","affiliation":[{"name":"Austrian Centre of Industrial Biotechnology, acib GmbH","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azra","family":"Kapetanovi\u0107","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]},{"name":"Austrian Centre of Industrial Biotechnology, acib GmbH","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Uma","family":"Isakovi\u0107","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanne","family":"Lux","sequence":"additional","affiliation":[{"name":"Institute of Chemical Engineering and Environmental Technology, Graz University of Technology","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sven","family":"Heimhilcher","sequence":"additional","affiliation":[{"name":"bisy GmbH","place":["Hofst\u00e4tten ander Raab, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Winkler","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anton","family":"Glieder","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anita","family":"Emmerstorfer-Augustin","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz","place":["Graz, Austria"]},{"name":"Austrian Centre of Industrial Biotechnology, acib GmbH","place":["Graz, Austria"]},{"name":"BioTechMed-Graz","place":["Graz, Austria"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"B2","doi-asserted-by":"publisher","first-page":"5301","DOI":"10.1007\/s00253-014-5732-5","article-title":"Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production","volume":"98","author":"Ahmad","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"441","DOI":"10.3390\/BIOM13030441","article-title":"Industrial production of proteins with Pichia pastoris-Komagataella phaffii","volume":"13","author":"Barone","year":"2023","journal-title":"Appl. Biomol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"3314","DOI":"10.1128\/AEM.02206-09","article-title":"Use of the yeast Pichia pastoris as an expression host for secretion of enterocin L50, a leaderless two-peptide (L50A and L50B) bacteriocin from Enterococcus faecium L50","volume":"76","author":"Basanta","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"607028","DOI":"10.3389\/fmicb.2020.607028","article-title":"Komagataella phaffii as emerging model organism in fundamental research","volume":"11","author":"Bernauer","year":"2021","journal-title":"Front. Microbiol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"1528","DOI":"10.1128\/mcb.00962-10","article-title":"Cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation depend on Cdc48 binding","volume":"31","author":"B\u00f6hm","year":"2011","journal-title":"Mol. Cell. Biol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"427","DOI":"10.1038\/NSB0698-427","article-title":"Solution structure of the thermostable sweet-tasting protein brazzein","volume":"5","author":"Caldwell","year":"1998","journal-title":"Nat. Struct. Biol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1186\/1471-2105-10-421","article-title":"BLAST+: architecture and applications","volume":"10","author":"Camacho","year":"2009","journal-title":"BMC Bioinforma."},{"key":"B9","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1016\/j.enzmictec.2004.05.007","article-title":"A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase","volume":"35","author":"Chang-Chih","year":"2004","journal-title":"Enzyme Microb. Technol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"13869","DOI":"10.1073\/pnas.1835918100","article-title":"Spermidine but not spermine is essential for hypusine biosynthesis and growth in Saccharomyces cerevisiae","volume":"100","author":"Chattopadhyay","year":"2003","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41598-017-15149-3","article-title":"High-level heterologous production and functional secretion by recombinant Pichia pastoris of the shortest proline-rich antibacterial honeybee peptide apidaecin","volume":"7","author":"Chen","year":"2017","journal-title":"Sci. Rep."},{"key":"B12","doi-asserted-by":"publisher","first-page":"10","DOI":"10.1016\/j.procbio.2017.07.006","article-title":"A novel strategy to prepare the precursor peptide of liraglutide","volume":"62","author":"Cheng","year":"2017","journal-title":"Process Biochem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"872","DOI":"10.1111\/1574-6976.12020","article-title":"The secretory pathway: exploring yeast diversity","volume":"37","author":"Delic","year":"2013","journal-title":"FEMS Microbiol. Rev."},{"key":"B14","doi-asserted-by":"publisher","first-page":"413","DOI":"10.3109\/10409238.2014.939608","article-title":"The hypusine-containing translation factor eIF5A","volume":"49","author":"Dever","year":"2014","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1186\/S12934-014-0125-0","article-title":"Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting","volume":"13","author":"Fitzgerald","year":"2014","journal-title":"Microb. Cell. Factories"},{"key":"B16","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"B17","doi-asserted-by":"publisher","first-page":"143","DOI":"10.1186\/s12934-026-02988-x","article-title":"Enhanced secretion of thermostable phytases from Myceliophthora thermophila by Komagataella phaffii","volume":"25","author":"Gratzer","year":"2026","journal-title":"Microb. Cell. Fact."},{"key":"B18","doi-asserted-by":"publisher","first-page":"876","DOI":"10.1002\/bab.2158","article-title":"Successful use of Nicotiana tabacum hairy roots for the recombinant production of Cecropin A peptide","volume":"69","author":"Hashemi","year":"2021","journal-title":"Biotechnol. Appl. Biochem."},{"key":"B19","doi-asserted-by":"publisher","first-page":"1356","DOI":"10.1099\/mic.0.000105","article-title":"Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization","volume":"161","author":"Heiss","year":"2015","journal-title":"Microbiology"},{"key":"B20","doi-asserted-by":"publisher","first-page":"1305","DOI":"10.1002\/YEA.320101007","article-title":"Rapid protein extraction from Saccharomyces cerevisiae","volume":"10","author":"Horvath","year":"1994","journal-title":"Yeast"},{"key":"B21","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s42003-022-03475-w","article-title":"A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii","volume":"5","author":"Ito","year":"2022","journal-title":"Commun. Biol."},{"key":"B22","doi-asserted-by":"publisher","first-page":"634","DOI":"10.1002\/IJCH.201600096","article-title":"Structural biology of calcitonin: from aqueous therapeutic properties to amyloid aggregation","volume":"57","author":"Kamgar-Parsi","year":"2017","journal-title":"Israel J. Chem."},{"key":"B23","doi-asserted-by":"publisher","first-page":"752742","DOI":"10.3389\/fmicb.2021.752742","article-title":"Overlapping roles of yeast transporters Aqr1, Qdr2, and Qdr3 in amino acid excretion and cross-feeding of lactic acid bacteria","volume":"12","author":"Kapetanakis","year":"2021","journal-title":"Front. Microbiol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"4319","DOI":"10.3390\/en14144319","article-title":"Catalytic hydrogenation of CO2 to methanol over Cu\/MgO catalysts in a semi-continuous reactor","volume":"14","author":"Kleiber","year":"2021","journal-title":"Energies"},{"key":"B25","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1002\/(SICI)1097-0282(199604)38:4<553::AID-BIP10>3.0.CO;2-B","article-title":"Assignment of the disulfide bonds in the sweet protein brazzein","volume":"38","author":"Kohmura","year":"1996","journal-title":"Biopolymers"},{"key":"B26","doi-asserted-by":"publisher","first-page":"22","DOI":"10.1186\/1475-2859-11-22","article-title":"Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway","volume":"11","author":"Krainer","year":"2012","journal-title":"Microb. Cell. Factories"},{"key":"B27","doi-asserted-by":"publisher","first-page":"2712","DOI":"10.1091\/MBC.E12-03-0228","article-title":"Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation","volume":"23","author":"Lakkaraju","year":"2012","journal-title":"Mol. Biol. Cell."},{"key":"B28","doi-asserted-by":"publisher","first-page":"1925","DOI":"10.1007\/S10529-013-1290-7","article-title":"Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins","volume":"35","author":"Larsen","year":"2013","journal-title":"Biotechnol. Lett."},{"key":"B29","doi-asserted-by":"publisher","first-page":"104888","DOI":"10.1016\/J.ISCI.2022.104888","article-title":"Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii","volume":"25","author":"Lehmayer","year":"2022","journal-title":"iScience"},{"key":"B30","doi-asserted-by":"publisher","first-page":"318","DOI":"10.1080\/21655979.2020.1736237","article-title":"Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris","volume":"11","author":"Li","year":"2020","journal-title":"Bioengineered"},{"key":"B31","doi-asserted-by":"publisher","first-page":"44","DOI":"10.2144\/05381BM04","article-title":"Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris","volume":"38","author":"Lin-Cereghino","year":"2005","journal-title":"BioTechniques"},{"key":"B32","doi-asserted-by":"publisher","first-page":"138","DOI":"10.1007\/s00253-025-13532-z","article-title":"The MF\u03b1 signal sequence in yeast-based protein secretion: challenges and innovations","volume":"109","author":"Merka\u0161","year":"2025","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"241","DOI":"10.1007\/978-1-0716-4779-0_16","article-title":"Cloning of MF\u03b1 secretion signal variants for protein secretion by Pichia pastoris","volume":"2697","author":"Merka\u0161","year":"2026","journal-title":"Methods Mol. Biol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1038\/s41573-020-00135-8","article-title":"Trends in peptide drug discovery","volume":"20","author":"Muttenthaler","year":"2021","journal-title":"Nat. Reviews-Drug Discov."},{"key":"B35","doi-asserted-by":"publisher","first-page":"e39720","DOI":"10.1371\/journal.pone.0039720","article-title":"Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology","volume":"7","author":"N\u00e4\u00e4tsaari","year":"2012","journal-title":"PLoS ONE"},{"key":"B36","doi-asserted-by":"publisher","first-page":"46","DOI":"10.3390\/life11010046","article-title":"Comparison of different signal peptides for the efficient secretion of the sweet-tasting plant protein brazzein in Pichia pastoris","volume":"11","author":"Neiers","year":"2021","journal-title":"Life"},{"key":"B37","doi-asserted-by":"publisher","first-page":"1719","DOI":"10.3390\/antibiotics12121719","article-title":"Bioengineering the antimicrobial activity of yeast by recombinant thanatin production","volume":"12","author":"Pipiya","year":"2023","journal-title":"Antibiotics"},{"key":"B38","doi-asserted-by":"publisher","first-page":"9807","DOI":"10.1021\/JF301600M","article-title":"Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris","volume":"60","author":"Poirier","year":"2012","journal-title":"J. Agric. Food Chem."},{"key":"B39","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1046\/J.1432-1327.1999.00749.X","article-title":"Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris","volume":"265","author":"Raemaekers","year":"1999","journal-title":"Eur. J. Biochem."},{"key":"B40","doi-asserted-by":"publisher","first-page":"122","DOI":"10.1186\/S13068-021-01971-5","article-title":"Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris","volume":"14","author":"Rieder","year":"2021","journal-title":"Biotechnol. Biofuels"},{"key":"B41","doi-asserted-by":"publisher","first-page":"e70046","DOI":"10.1002\/biot.70046","article-title":"Systematic engineering of Komagataella phaffii for efficient production of sweet-tasting protein brazzein","volume":"20","author":"Rong","year":"2025","journal-title":"Biotechnol. J."},{"key":"B42","doi-asserted-by":"publisher","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"B43","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1007\/978-1-59745-456-8_15","article-title":"Identification of pexophagy genes by restriction enzyme-mediated integration","volume":"389","author":"Schroder","year":"2007","journal-title":"Pichia Protocols- Methods Mol. Biol."},{"key":"B44","doi-asserted-by":"publisher","first-page":"e00296\u201322","DOI":"10.1128\/aem.00296-22","article-title":"Potential of the signal peptide derived from the PAS_chr3_0030 gene product for secretory expression of valuable enzymes in Pichia pastoris","volume":"88","author":"Shen","year":"2022","journal-title":"Appl. Environ. Microbiol."},{"key":"B45","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1038\/MSB.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"B46","doi-asserted-by":"publisher","first-page":"515","DOI":"10.1016\/j.tibs.2011.06.001","article-title":"Cdc48: a power machine in protein degradation","volume":"36","author":"Stolz","year":"2011","journal-title":"Trends Biochem. Sci."},{"key":"B47","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1016\/J.JBIOTEC.2016.04.023","article-title":"Refined Pichia pastoris reference genome sequence","volume":"235","author":"Sturmberger","year":"2016","journal-title":"J. Biotechnol."},{"key":"B48","doi-asserted-by":"publisher","first-page":"836","DOI":"10.1080\/07388551.2024.2412780","article-title":"Enhancing cyclotide bioproduction: harnessing biological synthesis methods and various expression systems for large-scale manufacturing","volume":"45","author":"Taghizadeh","year":"2025","journal-title":"Crit. Rev. Biotechnol."},{"key":"B49","doi-asserted-by":"publisher","first-page":"e0164771","DOI":"10.1371\/JOURNAL.PONE.0164771","article-title":"In vivo effects of Pichia pastoris-expressed antimicrobial peptide hepcidin on the community composition and metabolism of gut microbiota of rats","volume":"11","author":"Tian","year":"2016","journal-title":"PLoS ONE"},{"key":"B50","doi-asserted-by":"publisher","first-page":"1273","DOI":"10.1002\/elps.201800219","article-title":"Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins","volume":"40","author":"Tiwari","year":"2019","journal-title":"Electrophoresis"},{"key":"B51","doi-asserted-by":"publisher","first-page":"1492","DOI":"10.1128\/EC.3.6.1492-1503.2004","article-title":"Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretion of amino acids","volume":"3","author":"Velasco","year":"2004","journal-title":"Eukaryot. Cell."},{"key":"B52","doi-asserted-by":"publisher","first-page":"172","DOI":"10.1021\/acssynbio.5b00199","article-title":"A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris","volume":"5","author":"Vogl","year":"2016","journal-title":"ACS Synth. Biol."},{"key":"B53","doi-asserted-by":"publisher","first-page":"848","DOI":"10.1007\/s11814-010-0439-5","article-title":"Expression and purification of beefy meaty peptide in Pichia pastoris","volume":"28","author":"Wang","year":"2011","journal-title":"Korean J. Chem. Eng."},{"key":"B54","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1038\/s41392-022-00904-4","article-title":"Therapeutic peptides: current applications and future directions","volume":"7","author":"Wang","year":"2022","journal-title":"Signal Transduct. Target. Ther."},{"key":"B55","doi-asserted-by":"publisher","first-page":"3183","DOI":"10.1002\/jcb.26474","article-title":"Expanding the CRISPR\/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers","volume":"119","author":"Weninger","year":"2018","journal-title":"J. Cell. Biochem."},{"key":"B56","doi-asserted-by":"publisher","first-page":"1087","DOI":"10.1002\/(SICI)1097-0061(199908)15:11<1087::AID-YEA436>3.0.CO;2-F","article-title":"High-yield secretion of recombinant gelatins by Pichia pastoris","volume":"15","author":"Werten","year":"1999","journal-title":"Yeast"},{"key":"B57","doi-asserted-by":"publisher","first-page":"274","DOI":"10.1016\/J.SBI.2011.01.007","article-title":"The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling","volume":"21","author":"Wilson","year":"2011","journal-title":"Curr. Opin. Struct. Biol."},{"key":"B58","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1016\/S1046-5928(03)00048-2","article-title":"Overexpression of a small medicinal peptide from ginseng in the yeast Pichia pastoris","volume":"29","author":"Yan","year":"2003","journal-title":"Protein Expr. Purif."},{"key":"B59","doi-asserted-by":"publisher","first-page":"33697","DOI":"10.1074\/JBC.M113.508283","article-title":"Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae","volume":"288","author":"Zhang","year":"2013","journal-title":"J. Biol. Chem."},{"key":"B60","doi-asserted-by":"publisher","first-page":"681","DOI":"10.1007\/S00253-013-4881-2","article-title":"High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus","volume":"98","author":"Zhang","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."}],"container-title":["Frontiers in Bioengineering and Biotechnology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2026.1929744\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:30:43Z","timestamp":1789536643000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fbioe.2026.1929744\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,16]]},"references-count":60,"alternative-id":["10.3389\/fbioe.2026.1929744"],"URL":"https:\/\/doi.org\/10.3389\/fbioe.2026.1929744","relation":{},"ISSN":["2296-4185"],"issn-type":[{"value":"2296-4185","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,16]]},"article-number":"1929744"}}