{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T10:50:37Z","timestamp":1789469437555,"version":"build-2803163510"},"reference-count":56,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,1,30]],"date-time":"2026-01-30T00:00:00Z","timestamp":1769731200000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100002666","name":"Aalto University","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100002666","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Appl Microbiol Biotechnol"],"abstract":"<jats:sec>\n                    <jats:title>Abstract<\/jats:title>\n                    <jats:p>\n                      <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                      is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression:\n                      <jats:italic>PEP1<\/jats:italic>\n                      and\n                      <jats:italic>VPS30<\/jats:italic>\n                      , which both can channel proteins to the vacuole for degradation;\n                      <jats:italic>MON2<\/jats:italic>\n                      , which can lead to the re-uptake of secreted proteins; and\n                      <jats:italic>ALG3<\/jats:italic>\n                      , which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features\n                      <jats:italic>VPS30<\/jats:italic>\n                      ,\n                      <jats:italic>PEP1<\/jats:italic>\n                      , and\n                      <jats:italic>ALG3<\/jats:italic>\n                      deletions, and the small-scale fed-batch system, we obtained 2.5\u00a0\u00b5g\/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Chemical chaperones and amino acid-rich medium increased secreted protein titers.<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\u00a0\n                      <jats:italic>Medium and host engineering are instrumental for improving productivity.<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\u00a0\n                      <jats:italic>Small-scale cultivation system enables production levels suitable for characterization.<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-025-13700-1","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T17:24:36Z","timestamp":1769707476000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["An integrated cell and medium engineering approach for production of a nanobody fusion in Saccharomyces cerevisiae"],"prefix":"10.1007","volume":"110","author":[{"given":"Laura R. K.","family":"Niemel\u00e4","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lotta-Mari","family":"Kirjavainen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hendrikje C. J.","family":"Kozlowski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heidi","family":"Salminen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4371-0227","authenticated-orcid":false,"given":"Alexander D.","family":"Frey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,1,29]]},"reference":[{"key":"13700_CR1","doi-asserted-by":"publisher","first-page":"909","DOI":"10.1007\/s00726-015-1918-0","volume":"47","author":"Y Abe","year":"2015","unstructured":"Abe Y, Ohkuri T, Yoshitomi S, Murakami S, Ueda T (2015) Role of the osmolyte taurine on the folding of a model protein, hen egg white lysozyme, under a crowding condition. Amino Acids 47:909\u2013915. https:\/\/doi.org\/10.1007\/s00726-015-1918-0","journal-title":"Amino Acids"},{"key":"13700_CR2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.01589","volume":"8","author":"M Arbabi-Ghahroudi","year":"2017","unstructured":"Arbabi-Ghahroudi M (2017) Camelid single-domain antibodies: historical perspective and future outlook. Front Immunol 8:1589. https:\/\/doi.org\/10.3389\/fimmu.2017.01589","journal-title":"Front Immunol"},{"key":"13700_CR3","doi-asserted-by":"publisher","first-page":"97","DOI":"10.1016\/j.yexmp.2011.10.012","volume":"92","author":"P Ayala","year":"2012","unstructured":"Ayala P, Montenegro J, Vivar R, Letelier A, Urroz PA, Copaja M, Pivet D, Humeres C, Troncoso R, Vicencio JM, Lavandero S, D\u00edaz-Araya G (2012) Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol. Exp Mol Pathol 92:97\u2013104. https:\/\/doi.org\/10.1016\/j.yexmp.2011.10.012","journal-title":"Exp Mol Pathol"},{"key":"13700_CR4","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.01603","volume":"8","author":"P Bannas","year":"2017","unstructured":"Bannas P, Hambach J, Koch-Nolte F (2017) Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics. Front Immunol 8:1603. https:\/\/doi.org\/10.3389\/fimmu.2017.01603","journal-title":"Front Immunol"},{"key":"13700_CR5","doi-asserted-by":"publisher","DOI":"10.1186\/1475-2859-10-93","volume":"10","author":"K Baumann","year":"2011","unstructured":"Baumann K, Adelantado N, Lang C, Mattanovich D, Ferrer P (2011) Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris. Microb Cell Fact 10:93. https:\/\/doi.org\/10.1186\/1475-2859-10-93","journal-title":"Microb Cell Fact"},{"key":"13700_CR6","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1016\/j.canlet.2009.08.003","volume":"289","author":"A Bell","year":"2010","unstructured":"Bell A, Wang ZJ, Arbabi-Ghahroudi M, Chang TA, Durocher Y, Trojahn U, Baardsnes J, Jaramillo ML, Li S, Baral TN, O\u2019Connor-McCourt M, MacKenzie R, Zhang J (2010) Differential tumor-targeting abilities of three single-domain antibody formats. Cancer Lett 289:81\u201390. https:\/\/doi.org\/10.1016\/j.canlet.2009.08.003","journal-title":"Cancer Lett"},{"key":"13700_CR7","doi-asserted-by":"publisher","first-page":"5022","DOI":"10.1073\/pnas.0931263100","volume":"100","author":"B-KB-K Choi","year":"2003","unstructured":"Choi B-KB-K, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 100:5022\u20135027. https:\/\/doi.org\/10.1073\/pnas.0931263100","journal-title":"Proc Natl Acad Sci USA"},{"key":"13700_CR8","doi-asserted-by":"publisher","first-page":"9061","DOI":"10.1007\/s00253-015-6807-7","volume":"99","author":"JC de Ruijter","year":"2015","unstructured":"de Ruijter JC, Frey AD (2015) Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption. Appl Microbiol Biotechnol 99:9061\u20139071. https:\/\/doi.org\/10.1007\/s00253-015-6807-7","journal-title":"Appl Microbiol Biotechnol"},{"key":"13700_CR9","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1093\/femsyr\/fow104","volume":"17","author":"JC de Ruijter","year":"2016","unstructured":"de Ruijter JC, Jurgens G, Frey AD (2016a) Screening for novel genes of Saccharomyces cerevisiae involved in recombinant antibody production. FEMS Yeast Res 17:1\u201310. https:\/\/doi.org\/10.1093\/femsyr\/fow104","journal-title":"FEMS Yeast Res"},{"key":"13700_CR10","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-016-0488-5","volume":"15","author":"JC de Ruijter","year":"2016","unstructured":"de Ruijter JC, Koskela EV, Frey AD (2016b) Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum. Microb Cell Fact 15:87. https:\/\/doi.org\/10.1186\/s12934-016-0488-5","journal-title":"Microb Cell Fact"},{"key":"13700_CR11","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/yea.3298","volume":"35","author":"JC de Ruijter","year":"2017","unstructured":"de Ruijter JC, Koskela EV, Nandania J, Frey AD, Velagapudi V (2017) Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach. Yeast 35:1\u201311. https:\/\/doi.org\/10.1002\/yea.3298","journal-title":"Yeast"},{"key":"13700_CR12","doi-asserted-by":"publisher","first-page":"803","DOI":"10.1038\/nsb0996-803","volume":"3","author":"A Desmyter","year":"1996","unstructured":"Desmyter A, Transue T, Ghahroudi M, Thi M, Poortmans F, Hamers TR, Muyldermans S, Wyns L (1996) Crystal structure of a camel single domain VH antibody fragment in complex with lysozyme. Nat Struct Biol 3:803\u2013811. https:\/\/doi.org\/10.1038\/nsb0996-803","journal-title":"Nat Struct Biol"},{"key":"13700_CR13","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-014-0125-0","volume":"13","author":"I Fitzgerald","year":"2014","unstructured":"Fitzgerald I, Glick BS (2014) Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting. Microb Cell Fact 13:125. https:\/\/doi.org\/10.1186\/s12934-014-0125-0","journal-title":"Microb Cell Fact"},{"key":"13700_CR14","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1016\/S1097-2765(00)80017-9","volume":"1","author":"AR Frand","year":"1998","unstructured":"Frand AR, Kaiser CA (1998) The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum. Mol Cell 1:161\u2013170","journal-title":"Mol Cell"},{"key":"13700_CR15","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/S0168-1656(99)00228-X","volume":"78","author":"LGJ Frenken","year":"2000","unstructured":"Frenken LGJ, Van Der Linden RHJ, Hermans PWJJ, Wil Bos J, Ruuls RC, De Geus B, Verrips CT (2000) Isolation of antigen specific Llama VHH antibody fragments and their high-level secretion by Saccharomyces cerevisiae. J Biotechnol 78:11\u201321","journal-title":"J Biotechnol"},{"key":"13700_CR16","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1007\/s10529-006-9237-x","volume":"29","author":"B Gasser","year":"2007","unstructured":"Gasser B, Mattanovich D (2007) Antibody production with yeasts and filamentous fungi: on the road to large scale? Biotechnol Lett 29:201\u2013212","journal-title":"Biotechnol Lett"},{"key":"13700_CR17","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1038\/nprot.2007.15","volume":"2","author":"R Gietz","year":"2007","unstructured":"Gietz R, Schiestl R (2007) Large-scale high-efficiency yeast transformation using the LiAc\/SS carrier DNA\/PEG method. Nat Protoc 2:38\u201341","journal-title":"Nat Protoc"},{"key":"13700_CR18","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1093\/protein\/gzr057","volume":"25","author":"A Gorlani","year":"2012","unstructured":"Gorlani A, Lutje Hulsik D, Adams H, Vriend G, Hermans P, Verrips T (2012) Antibody engineering reveals the important role of J segments in the production efficiency of llama single-domain antibodies in Saccharomyces cerevisiae. Protein Eng des Sel 25:39\u201346. https:\/\/doi.org\/10.1093\/protein\/gzr057","journal-title":"Protein Eng des Sel"},{"key":"13700_CR19","doi-asserted-by":"publisher","first-page":"2519","DOI":"10.1093\/nar\/24.13.2519","volume":"24","author":"U G\u00fcldener","year":"1996","unstructured":"G\u00fcldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519\u20132524. https:\/\/doi.org\/10.1093\/nar\/24.13.2519","journal-title":"Nucleic Acids Res"},{"key":"13700_CR20","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1038\/363446a0","volume":"336","author":"C Hamers-Casterman","year":"1993","unstructured":"Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Bajyana Songa E, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 336:446\u2013448. https:\/\/doi.org\/10.1038\/363446a0","journal-title":"Nature"},{"key":"13700_CR21","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1007\/978-1-61779-197-0_12","volume":"765","author":"JH Hegemann","year":"2011","unstructured":"Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. Methods Mol Biol 765:189\u2013206","journal-title":"Methods Mol Biol"},{"key":"13700_CR22","doi-asserted-by":"publisher","first-page":"8547","DOI":"10.1007\/s00253-012-4639-2","volume":"97","author":"X Ji","year":"2013","unstructured":"Ji X, Lu W, Zhou H, Han D, Yang L, Wu H, Li J, Liu H, Zhang J, Cao P, Zhang S (2013) Covalently dimerized Camelidae antihuman TNFa single-domain antibodies expressed in yeast Pichia pastoris show superior neutralizing activity. Appl Microbiol Biotechnol 97:8547\u20138558. https:\/\/doi.org\/10.1007\/s00253-012-4639-2","journal-title":"Appl Microbiol Biotechnol"},{"key":"13700_CR23","doi-asserted-by":"publisher","DOI":"10.3390\/ijms24065994","volume":"24","author":"BK Jin","year":"2023","unstructured":"Jin BK, Odongo S, Radwanska M, Magez S (2023) A review of diagnostic and therapeutic applications. Int J Mol Sci 24:5994. https:\/\/doi.org\/10.3390\/ijms24065994","journal-title":"Int J Mol Sci"},{"key":"13700_CR24","doi-asserted-by":"publisher","first-page":"4914","DOI":"10.1128\/mcb.22.13.4914-4928.2002","volume":"22","author":"A Jochum","year":"2002","unstructured":"Jochum A, Jackson D, Schwarz H, Pipkorn R, Singer-Kr\u00fcger B (2002) Yeast Ysl2p, homologous to Sec7 domain guanine nucleotide exchange factors, functions in endocytosis and maintenance of vacuole integrity and interacts with the Arf-like small GTPase Arl1p. Mol Cell Biol 22:4914\u20134928. https:\/\/doi.org\/10.1128\/mcb.22.13.4914-4928.2002","journal-title":"Mol Cell Biol"},{"key":"13700_CR25","doi-asserted-by":"publisher","first-page":"2527","DOI":"10.1002\/bit.25687","volume":"112","author":"YB Johari","year":"2015","unstructured":"Johari YB, Estes SD, Alves CS, Sinacore MS, James DC (2015) Integrated cell and process engineering for improved transient production of a \u201cdifficult-to-express\u201d fusion protein by CHO cells. Biotechnol Bioeng 112:2527\u20132542. https:\/\/doi.org\/10.1002\/bit.25687","journal-title":"Biotechnol Bioeng"},{"key":"13700_CR26","doi-asserted-by":"publisher","DOI":"10.1186\/1475-2859-2-1","volume":"2","author":"V Joosten","year":"2003","unstructured":"Joosten V, Lokman C, Van Den Hondel CA, Punt PJ (2003) The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. Microb Cell Fact 2:1. https:\/\/doi.org\/10.1186\/1475-2859-2-1","journal-title":"Microb Cell Fact"},{"key":"13700_CR27","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1007\/s002530051659","volume":"53","author":"HA Kang","year":"2000","unstructured":"Kang HA, Choi E-S, Hong W-K, Kim J-Y, Ko S-M, Sohn J-H, Rhee SK (2000) Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:575\u2013582","journal-title":"Appl Microbiol Biotechnol"},{"key":"13700_CR28","doi-asserted-by":"publisher","DOI":"10.1093\/femsyr\/foy016","volume":"18","author":"CT Mai","year":"2018","unstructured":"Mai CT, Le QG, Ishiwata-Kimata Y, Takagi H, Kohno K, Kimata Y (2018) 4-phenylbutyrate suppresses the unfolded protein response without restoring protein folding in Saccharomyces cerevisiae. FEMS Yeast Res 18:foy016. https:\/\/doi.org\/10.1093\/femsyr\/foy016","journal-title":"FEMS Yeast Res"},{"key":"13700_CR29","doi-asserted-by":"publisher","first-page":"5767","DOI":"10.1093\/nar\/22.25.5767","volume":"22","author":"D Mumberg","year":"1994","unstructured":"Mumberg D, Muller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22:5767\u20135768. https:\/\/doi.org\/10.1093\/nar\/22.25.5767","journal-title":"Nucleic Acids Res"},{"key":"13700_CR30","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","volume":"82","author":"S Muyldermans","year":"2013","unstructured":"Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775\u2013797. https:\/\/doi.org\/10.1146\/annurev-biochem-063011-092449","journal-title":"Annu Rev Biochem"},{"key":"13700_CR31","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jbiotec.2024.03.009","volume":"387","author":"LRK Niemel\u00e4","year":"2024","unstructured":"Niemel\u00e4 LRK, Koskela EV, Frey AD (2024) Modification of the endoplasmic reticulum morphology enables improved recombinant antibody expression in Saccharomyces cerevisiae. J Biotechnol 387:1\u201311. https:\/\/doi.org\/10.1016\/j.jbiotec.2024.03.009","journal-title":"J Biotechnol"},{"key":"13700_CR32","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1093\/femsyr\/foaa002","volume":"20","author":"MA Piirainen","year":"2020","unstructured":"Piirainen MA, Frey AD (2020) Investigating the role of ERAD on antibody processing in glycoengineered Saccharomyces cerevisiae. FEMS Yeast Res 20:1\u201311. https:\/\/doi.org\/10.1093\/femsyr\/foaa002","journal-title":"FEMS Yeast Res"},{"key":"13700_CR33","doi-asserted-by":"publisher","first-page":"532","DOI":"10.1016\/j.nbt.2014.03.001","volume":"31","author":"MA Piirainen","year":"2014","unstructured":"Piirainen MA, de Ruijter JC, Koskela EV, Frey AD (2014) Glycoengineering of yeasts from the perspective of glycosylation efficiency. N Biotechnol 31:532\u2013537. https:\/\/doi.org\/10.1016\/j.nbt.2014.03.001","journal-title":"N Biotechnol"},{"key":"13700_CR34","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1007\/s00253-021-11727-8","volume":"106","author":"MA Piirainen","year":"2021","unstructured":"Piirainen MA, Salminen H, Frey AD (2021) Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae. Appl Microbiol Biotechnol 106:301\u2013315. https:\/\/doi.org\/10.1007\/s00253-021-11727-8","journal-title":"Appl Microbiol Biotechnol"},{"key":"13700_CR35","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/S0022-1759(00)00290-8","volume":"251","author":"DB Powers","year":"2001","unstructured":"Powers DB, Amersdorfer P, Poul M-A, Nielsen UB, Shalaby MR, Adams GP, Weiner LM, Marks JD (2001) Expression of single-chain Fv-Fc fusions in Pichia pastoris. J Immunol Methods 251:123\u2013135. https:\/\/doi.org\/10.1016\/S0022-1759(00)00290-8","journal-title":"J Immunol Methods"},{"key":"13700_CR36","doi-asserted-by":"publisher","first-page":"460","DOI":"10.1016\/j.coi.2008.06.012","volume":"20","author":"LG Presta","year":"2008","unstructured":"Presta LG (2008) Molecular engineering and design of therapeutic antibodies. Curr Opin Immunol 20:460\u2013470. https:\/\/doi.org\/10.1016\/j.coi.2008.06.012","journal-title":"Curr Opin Immunol"},{"key":"13700_CR37","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.pep.2016.03.004","volume":"123","author":"M Qasemi","year":"2016","unstructured":"Qasemi M, Behdani M, Shokrgozar MA, Molla-Kazemiha V, Mohseni-Kuchesfahani H, Habibi-Anbouhi M (2016) Construction and expression of an anti-VEGFR2 Nanobody-Fc fusionbody in NS0 host cell. Protein Expr Purif 123:19\u201325. https:\/\/doi.org\/10.1016\/j.pep.2016.03.004","journal-title":"Protein Expr Purif"},{"key":"13700_CR38","doi-asserted-by":"publisher","first-page":"1192","DOI":"10.1002\/bit.22338","volume":"103","author":"JA Rakestraw","year":"2009","unstructured":"Rakestraw JA, Sazinsky SL, Piatesi A, Antipov E, Wittrup KD (2009) Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae. Biotechnol Bioeng 103:1192\u20131201. https:\/\/doi.org\/10.1002\/bit.22338","journal-title":"Biotechnol Bioeng"},{"key":"13700_CR39","doi-asserted-by":"publisher","first-page":"376","DOI":"10.1002\/bit.25360","volume":"112","author":"WA Rodr\u00edguez-Limas","year":"2015","unstructured":"Rodr\u00edguez-Limas WA, Tannenbaum V, Tyo KEJ (2015) Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae. Biotechnol Bioeng 112:376\u2013385. https:\/\/doi.org\/10.1002\/bit.25360","journal-title":"Biotechnol Bioeng"},{"key":"13700_CR40","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0044505","volume":"7","author":"SD Roth","year":"2012","unstructured":"Roth SD, Sch\u00fcttrumpf J, Milanov P, Abriss D, Ungerer C, Quade-Lyssy P, Simpson JC, Pepperkok R, Seifried E, Tonn T (2012) Chemical chaperones improve protein secretion and rescue mutant factor VIII in mice with hemophilia A. PLoS ONE 7:e44505. https:\/\/doi.org\/10.1371\/journal.pone.0044505","journal-title":"PLoS ONE"},{"key":"13700_CR41","doi-asserted-by":"publisher","first-page":"695","DOI":"10.1016\/j.nucmedbio.2015.03.003","volume":"42","author":"M Rotman","year":"2015","unstructured":"Rotman M, Welling MM, van den Boogaard ML, Moursel LG, van der Graaf LM, van Buchem MA, van der Maarel SM, van der Weerd L (2015) Fusion of hIgG1-Fcto 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP\/PS1 mice but does not increase brain uptake. Nucl Med Biol 42:695\u2013702. https:\/\/doi.org\/10.1016\/j.nucmedbio.2015.03.003","journal-title":"Nucl Med Biol"},{"issue":"621","key":"13700_CR42","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.abi7826","volume":"13","author":"B Schepens","year":"2021","unstructured":"Schepens B, van Schie L, Nerinckx W, Roose K, Van Breedam W, Fijalkowska D, Devos S, Weyts W, De Cae S, Vanmarcke S, Lonigro C, Eeckhaut H, Van Herpe D, Borloo J, Oliveira AF, Catani JPP, Creytens S, De Vlieger D, Michielsen G, Marchan JCZ, Moschonas GD, Rossey I, Sedeyn K, Van Hecke A, Zhang X, Langendries L, Jacobs S, Ter Horst S, Seldeslachts L, Liesenborghs L, Boudewijns R, Thibaut HJ, Dallmeier K, Velde GV, Weynand B, Beer J, Schnepf D, Ohnemus A, Remory I, Foo CS, Abdelnabi R, Maes P, Kaptein SJF, Rocha-Pereira J, Jochmans D, Delang L, Peelman F, Staeheli P, Schwemmle M, Devoogdt N, Tersago D, Germani M, Heads J, Henry A, Popplewell A, Ellis M, Brady K, Turner A, Dombrecht B, Stortelers C, Neyts J, Callewaert N, Saelens X (2021) An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models. Sci Transl Med 13(621):eabi7826. https:\/\/doi.org\/10.1126\/scitranslmed.abi7826","journal-title":"Sci Transl Med"},{"key":"13700_CR43","doi-asserted-by":"publisher","first-page":"773","DOI":"10.1038\/nbt0898-773","volume":"16","author":"EV Shusta","year":"1998","unstructured":"Shusta EV, Raines RT, Pl\u00fcckthun A, Wittrup KD, Pluckthun A, Wittrup KD (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol 16:773\u2013777. https:\/\/doi.org\/10.1038\/nbt0898-773","journal-title":"Nat Biotechnol"},{"key":"13700_CR44","doi-asserted-by":"publisher","first-page":"13997","DOI":"10.1073\/pnas.0606236103","volume":"103","author":"TO Street","year":"2006","unstructured":"Street TO, Wayne Bolen D, Rose GD, Jenkins TC (2006) A molecular mechanism for osmolyte-induced protein stability. Proc Natl Acad Sci U S A 103:13997\u201314002","journal-title":"Proc Natl Acad Sci U S A"},{"key":"13700_CR45","doi-asserted-by":"publisher","first-page":"273","DOI":"10.1016\/S0141-0229(01)00497-5","volume":"30","author":"YE Thomassen","year":"2002","unstructured":"Thomassen YE, Meijer W, Sierkstra L, Verrips CT (2002) Large-scale production of VHH antibody fragments by Saccharomyces cerevisiae. Enzyme Microb Technol 30:273\u2013278. https:\/\/doi.org\/10.1016\/S0141-0229(01)00497-5","journal-title":"Enzyme Microb Technol"},{"key":"13700_CR46","doi-asserted-by":"publisher","first-page":"7149","DOI":"10.1007\/s00253-014-5783-7","volume":"98","author":"KEJ Tyo","year":"2014","unstructured":"Tyo KEJ, Liu Z, Magnusson Y, Petranovic D, Nielsen J (2014) Impact of protein uptake and degradation on recombinant protein secretion in yeast. Appl Microbiol Biotechnol 98:7149\u20137159. https:\/\/doi.org\/10.1007\/s00253-014-5783-7","journal-title":"Appl Microbiol Biotechnol"},{"key":"13700_CR47","doi-asserted-by":"publisher","first-page":"1722","DOI":"10.1038\/s41587-022-01582-x","volume":"40","author":"G Walsh","year":"2022","unstructured":"Walsh G, Walsh E (2022) Biopharmaceutical benchmarks 2022. Nat Biotechnol 40:1722\u20131760. https:\/\/doi.org\/10.1038\/s41587-022-01582-x","journal-title":"Nat Biotechnol"},{"key":"13700_CR48","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.copbio.2017.03.017","volume":"48","author":"G Wang","year":"2017","unstructured":"Wang G, Huang M, Nielsen J (2017) Exploring the potential of Saccharomyces cerevisiae for biopharmaceutical protein production. Curr Opin Biotechnol 48:77\u201384. https:\/\/doi.org\/10.1016\/j.copbio.2017.03.017","journal-title":"Curr Opin Biotechnol"},{"key":"13700_CR49","doi-asserted-by":"publisher","unstructured":"Wang Y, Li X, Chen X, Nielsen J, Petranovic D, Siewers V (2021) Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion. Microb Cell Fact. https:\/\/doi.org\/10.1186\/s12934-021-01624-0","DOI":"10.1186\/s12934-021-01624-0"},{"key":"13700_CR50","doi-asserted-by":"publisher","first-page":"731","DOI":"10.1126\/science.abc7424","volume":"369","author":"AZ Wec","year":"2020","unstructured":"Wec AZ, Wrapp D, Herbert AS, Maurer DP, Haslwanter D, Sakharkar M, Jangra RK, Dieterle ME, Lilov A, Huang D, Tse LV, Johnson NV, Hsieh C-L, Wang N, Nett JH, Champney E, Burnina I, Brown M, Lin S, Sinclair M, Johnson C, Pudi S, Iii RB, Wirchnianski AS, Laudermilch E, Florez C, Fels JM, O\u2019brien CM, Graham BS, Nemazee D, Burton DR, Baric RS, Voss JE, Chandran K, Dye JM, Mclellan JS, Walker LM (2020) Broad neutralization of SARS-related viruses by human monoclonal antibodies. Science 369:731\u2013736. https:\/\/doi.org\/10.1126\/science.abc7424","journal-title":"Science"},{"key":"13700_CR51","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1379\/1466-1268(1996)001<0109:IOMACC>2.3.CO;2","volume":"1","author":"W Welch","year":"1996","unstructured":"Welch W, Brown C (1996) Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1:109\u2013115","journal-title":"Cell Stress Chaperones"},{"key":"13700_CR52","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1002\/bit.22376","volume":"104","author":"P Xu","year":"2009","unstructured":"Xu P, Robinson AS (2009) Decreased secretion and unfolded protein response up-regulation are correlated with intracellular retention for single-chain antibody variants produced in yeast. Biotechnol Bioeng 104:20\u201329. https:\/\/doi.org\/10.1002\/bit.22376","journal-title":"Biotechnol Bioeng"},{"key":"13700_CR53","doi-asserted-by":"publisher","first-page":"269","DOI":"10.1016\/j.ymben.2005.04.002","volume":"7","author":"P Xu","year":"2005","unstructured":"Xu P, Raden D, Doyle FJ, Robinson AS (2005) Analysis of unfolded protein response during single-chain antibody expression in Saccharomyces cerevisiae reveals different roles for BiP and PDI in folding. Metab Eng 7:269\u2013279. https:\/\/doi.org\/10.1016\/j.ymben.2005.04.002","journal-title":"Metab Eng"},{"key":"13700_CR54","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-016-0575-7","volume":"15","author":"S Xu","year":"2016","unstructured":"Xu S, Zhang G-YY, Zhang H, Kitajima T, Nakanishi H, Gao X-DD (2016) Effects of Rho1, a small GTPase on the production of recombinant glycoproteins in Saccharomyces cerevisiae. Microb Cell Fact 15:179. https:\/\/doi.org\/10.1186\/s12934-016-0575-7","journal-title":"Microb Cell Fact"},{"key":"13700_CR55","doi-asserted-by":"publisher","DOI":"10.1016\/j.engmic.2023.100122","volume":"4","author":"S Yang","year":"2024","unstructured":"Yang S, Song L, Wang J, Zhao J, Tang H, Bao X (2024) Engineering Saccharomyces cerevisiae for efficient production of recombinant proteins. Environ Microbiol 4:100122. https:\/\/doi.org\/10.1016\/j.engmic.2023.100122","journal-title":"Environ Microbiol"},{"key":"13700_CR56","doi-asserted-by":"publisher","DOI":"10.1186\/s13567-020-00868-9","volume":"52","author":"L Zhang","year":"2021","unstructured":"Zhang L, Wang L, Cao S, Lv H, Huang J, Zhang G, Tabynov K, Zao Q, Zhou EN (2021) Nanobody Nb6 fused with porcine IgG Fcas the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages. Vet Res 52:25. https:\/\/doi.org\/10.1186\/s13567-020-00868-9","journal-title":"Vet Res"}],"container-title":["Applied Microbiology and Biotechnology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00253-025-13700-1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-025-13700-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-025-13700-1.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,30]],"date-time":"2026-01-30T07:35:51Z","timestamp":1769758551000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00253-025-13700-1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,29]]},"references-count":56,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["13700"],"URL":"https:\/\/doi.org\/10.1007\/s00253-025-13700-1","relation":{},"ISSN":["1432-0614"],"issn-type":[{"value":"1432-0614","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,29]]},"assertion":[{"value":"14 June 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"3 December 2025","order":2,"name":"revised","label":"Revised","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 December 2025","order":3,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"29 January 2026","order":4,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This article does not contain any studies with human participants or animals performed by any of the authors.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethical approval"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"46"}}