{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T13:30:55Z","timestamp":1788874255143,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,15]],"date-time":"2026-02-15T00:00:00Z","timestamp":1771113600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2021YFA0910602"],"award-info":[{"award-number":["2021YFA0910602"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences","award":["CAAS-BRC-LP-2025-02"],"award-info":[{"award-number":["CAAS-BRC-LP-2025-02"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32171806"],"award-info":[{"award-number":["32171806"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Foods"],"abstract":"<jats:p>Bovine chymosin is key for cheese production, yet its traditional sourcing is unsustainable. While microbial and plant-based alternatives exist, they often cause non-specific proteolysis, leading to bitter flavors in cheese. This study aims to develop a high-yield, methanol-independent platform for recombinant bovine chymosin production by engineering the expression system of Komagataella phaffii through multi-factorial optimization. Initially, the native bovine prochymosin gene (pcw) was codon-optimized (pcm14) and cloned, along with an mCherry-tag construct (clpcm14), into inducible vector pPIC9 for expression in Komagataella phaffii GS115. Screening identified the fusion-tagged strain clp2-91 as the highest producer. Subsequently, the inducible AOX1 promoter in the previously selected clp2-91 strain was replaced with a constitutive GAP promoter, yielding engineered strain GH1. Cultivated in a 3L fermenter, GH1 exhibited a volumetric productivity of 105.03 SU\/(mL\u00b7h), twice that of inducible strain clp2-91 (53.59 SU\/(mL\u00b7h)). The further optimization of fermentation conditions (pH 4.0, glucose as carbon source, fed-batch mode) boosted the enzyme activity of GH1 to 12,000 SU\/mL. The recombinant chymosin exhibited enzymatic properties similar to those of the native enzyme and, importantly, demonstrated a broader pH stability (pH 2.0\u20136.0). This study demonstrates an efficient strategy for chymosin expression in K. phaffii, offering insights that may support the future development and optimization of heterologous protein production in this yeast.<\/jats:p>","DOI":"10.3390\/foods15040731","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T14:55:47Z","timestamp":1771253747000},"page":"731","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Boosting Recombinant Bovine Chymosin in Komagataella phaffii via Fusion Protein and Constitutive Promoter Expression"],"prefix":"10.3390","volume":"15","author":[{"given":"Xinrun","family":"Ren","sequence":"first","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"},{"name":"College of Forestry, Shanxi Agricultural University, Jinzhong 030801, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyan","family":"Ning","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Liu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4060-4517","authenticated-orcid":false,"given":"Xinxin","family":"Xu","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lina","family":"Men","sequence":"additional","affiliation":[{"name":"College of Forestry, Shanxi Agricultural University, Jinzhong 030801, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3954-6911","authenticated-orcid":false,"given":"Angie","family":"Deng","sequence":"additional","affiliation":[{"name":"Lucile Packard Children\u2019s Hospital at Stanford, Palo Alto, CA 94303, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5415-1443","authenticated-orcid":false,"given":"Yuhong","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1873-5617","authenticated-orcid":false,"given":"Zhiwei","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Forestry, Shanxi Agricultural University, Jinzhong 030801, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Key Laboratory of Agricultural Microbiology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Han, Y., Zhang, L.Q., Rao, D.M., Lei, L., and Yang, J.K. (2025). Multiple strategies were adopted to optimize the enzymatic characteristics and improve the expression of bovine chymosin BtChy in Kluyveromyces lactis for cheese production. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1605229"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1134\/S0003683820040031","article-title":"Basic Biochemical Properties of Recombinant Chymosins (Review)","volume":"56","author":"Belenkaya","year":"2020","journal-title":"Appl. Biochem. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"105179","DOI":"10.1016\/j.idairyj.2021.105179","article-title":"Rennet coagulation of heated milk: A review","volume":"124","author":"Britten","year":"2022","journal-title":"Int. Dairy J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"105796","DOI":"10.1016\/j.idairyj.2023.105796","article-title":"A novel approach to isolation of \u03b2-casein from micellar casein concentrate by cold microfiltration combined with chymosin treatment","volume":"148","author":"Goulding","year":"2024","journal-title":"Int. Dairy J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9952","DOI":"10.3168\/jds.2017-12979","article-title":"A 100-Year Review: Cheese production and quality","volume":"100","author":"Johnson","year":"2017","journal-title":"J. Dairy Sci."},{"key":"ref_6","first-page":"585","article-title":"Influence of diet and age of kids on enzymatic activities of kid rennet pastes","volume":"15","author":"Rossano","year":"2003","journal-title":"Ital. J. Food Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1111\/j.1471-0307.2010.00633.x","article-title":"Recent advances in milk clotting enzymes","volume":"64","author":"Jacob","year":"2011","journal-title":"Int. J. Dairy Technol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.ijbiomac.2018.10.006","article-title":"A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization","volume":"121","author":"Afsharnezhad","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Sbhatu, D.B., Tekle, H.T., and Tesfamariam, K.H. (2020). Ficus palmata Forsk\u00e5l (beles adgi) as a source of milk clotting agent: A preliminary research. BMC Res. Notes, 13.","DOI":"10.1186\/s13104-020-05293-x"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1080\/10826068.2019.1683861","article-title":"Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus","volume":"50","author":"Souto","year":"2020","journal-title":"Prep. Biochem. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.5713\/ajas.16.0038","article-title":"Cloning and Expression of Yak Active Chymosin in Pichia pastoris","volume":"29","author":"Luo","year":"2016","journal-title":"Asian-Australas. J. Anim. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"100094","DOI":"10.1016\/j.engmic.2023.100094","article-title":"Genetic tools for metabolic engineering of Pichia pastoris","volume":"3","author":"Wu","year":"2023","journal-title":"Eng. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13000","DOI":"10.1093\/nar\/gkaa1066","article-title":"Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression","volume":"48","author":"Ito","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8845","DOI":"10.1039\/D3GC02874G","article-title":"Development of high methanol-tolerance Pichia pastoris based on iterative adaptive laboratory evolution","volume":"25","author":"Wang","year":"2023","journal-title":"Green Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.yrtph.2006.02.001","article-title":"Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil","volume":"45","author":"Ciofalo","year":"2006","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.jare.2024.08.008","article-title":"Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris","volume":"73","author":"Zhang","year":"2025","journal-title":"J. Adv. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"109589","DOI":"10.1016\/j.enzmictec.2020.109589","article-title":"Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris","volume":"139","year":"2020","journal-title":"Enzyme Microb. Technol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1007\/s12033-016-9965-7","article-title":"Codon Optimization of the \u201cBos Taurus Chymosin\u201d Gene for the Production of Recombinant Chymosin in Pichia pastoris","volume":"58","author":"Sepulveda","year":"2016","journal-title":"Mol. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jbiotec.2024.04.008","article-title":"Optimizing Pichia pastoris protein secretion: Role of N-linked glycosylation on the \u03b1-mating factor secretion signal leader","volume":"391","author":"Dai","year":"2024","journal-title":"J. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2411","DOI":"10.1007\/s00253-020-10402-8","article-title":"Fusion tags to enhance heterologous protein expression","volume":"104","author":"Ki","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.pep.2015.03.012","article-title":"Expression and characterization of camel chymosin in Pichia pastoris","volume":"111","author":"Wang","year":"2015","journal-title":"Protein Expr. Purif."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1007\/s13205-024-04039-x","article-title":"A self-inducible heterologous protein expression system in Komagataella phaffii (Pichia pastoris)","volume":"14","author":"Unver","year":"2024","journal-title":"3 Biotech"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"41850","DOI":"10.1038\/srep41850","article-title":"Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris","volume":"7","author":"Wang","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1186\/s12934-024-02435-9","article-title":"Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins","volume":"23","author":"Lin","year":"2024","journal-title":"Microb. Cell Factories"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e07137","DOI":"10.1016\/j.heliyon.2021.e07137","article-title":"Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris","volume":"7","author":"Akishev","year":"2021","journal-title":"Heliyon"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1007\/s12033-023-00803-1","article-title":"A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production","volume":"66","author":"Vijayakumar","year":"2024","journal-title":"Mol. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1007\/s12033-020-00249-9","article-title":"mCherry Protein as an In Vivo Quantitative Reporter of Gene Expression in the Chloroplast of Chlamydomonas reinhardtii","volume":"62","author":"Kim","year":"2020","journal-title":"Mol. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1186\/s13068-022-02243-6","article-title":"The \u03b1-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii","volume":"15","author":"Zou","year":"2022","journal-title":"Biotechnol. Biofuels Bioprod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1016\/j.gene.2013.01.062","article-title":"The effect of \u03b1-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris","volume":"519","author":"Stark","year":"2013","journal-title":"Gene"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1111\/j.1432-1033.1979.tb12927.x","article-title":"Investigations on the activation of bovine prochymosin","volume":"94","author":"Pedersen","year":"1979","journal-title":"Eur. J. Biochem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/0076-6879(70)19033-1","article-title":"[30] Milk-clotting enzyme from Mucor pusillus var. Lindt","volume":"19","author":"Arima","year":"1970","journal-title":"Methods Enzymol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"254","DOI":"10.3923\/ijds.2017.254.265","article-title":"Purification and characterization of milk clotting enzyme from artichoke (Cynara cardunculus L.) flowers as coagulant on white soft cheese","volume":"12","author":"Ibrahim","year":"2017","journal-title":"Int. J. Dairy Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"138","DOI":"10.3923\/jbs.2020.138.146","article-title":"Enzymic milk clotting activities of metalloproteinase kistomin","volume":"20","author":"Vejayan","year":"2020","journal-title":"J. Biol. Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"132116","DOI":"10.1016\/j.biortech.2025.132116","article-title":"Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering","volume":"419","author":"Zheng","year":"2025","journal-title":"Bioresour. Technol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1016\/j.jfoodeng.2011.09.009","article-title":"Comparison of total milk-clotting activity measurement precision using the Berridge clotting time method and a proposed optical method","volume":"108","author":"Tabayehnejad","year":"2012","journal-title":"J. Food Eng."},{"key":"ref_36","unstructured":"(2007). Milk\u2014Determination of Total Milk-Clotting Activity of Bovine Rennets (Including Fermentation-Produced Chymosin) (Standard No. ISO 11815:2007)."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1186\/s12934-021-01580-9","article-title":"Codon usage bias regulates gene expression and protein conformation in yeast expression system P. pastoris","volume":"20","author":"Xu","year":"2021","journal-title":"Microb. Cell Fact."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"D\u00e4lken, B., Jabulowsky, R.A., Oberoi, P., Benhar, I., and Wels, W.S. (2010). Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0014404"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5893","DOI":"10.1007\/s00253-022-12139-y","article-title":"Pathway engineering facilitates efficient protein expression in Pichia pastoris","volume":"106","author":"Liu","year":"2022","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/bs.mie.2021.07.004","article-title":"Chapter Three\u2014Expression of proteins in Pichia pastoris","volume":"Volume 660","author":"Kelman","year":"2021","journal-title":"Methods in Enzymology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"S13","DOI":"10.1186\/1475-2859-5-S1-S13","article-title":"Substrate feeding strategies in Pichia pastoris fed-batch cultivation processes: Analysis of key parameters influencing recombinant protein production","volume":"5","author":"Ramon","year":"2006","journal-title":"Microb. Cell Factories"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"foab057","DOI":"10.1093\/femsyr\/foab057","article-title":"Recombinant protein production in Pichia pastoris: From transcriptionally redesigned strains to bioprocess optimization and metabolic modelling","volume":"21","author":"Berrios","year":"2021","journal-title":"FEMS Yeast Res."}],"container-title":["Foods"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2304-8158\/15\/4\/731\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T13:58:50Z","timestamp":1773064730000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2304-8158\/15\/4\/731"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,15]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["foods15040731"],"URL":"https:\/\/doi.org\/10.3390\/foods15040731","relation":{},"ISSN":["2304-8158"],"issn-type":[{"value":"2304-8158","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,15]]}}}