{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T04:58:48Z","timestamp":1790744328530,"version":"4.1.0"},"reference-count":20,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["mLife"],"abstract":"<jats:title>Impact statement<\/jats:title>\n                  <jats:p>\n                    Nonconventional yeast\n                    <jats:italic>Pichia kudriavzevii<\/jats:italic>\n                    possesses attractive phenotypic traits for industrial biomanufacturing; however, it remains genetically intractable compared with established yeast chassis. Here, we present an integrated synthetic biology framework that renders\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    a programmable production host. We established a highly efficient CRISPR\/Cas9 genome editing system in this diploid yeast, enabling marker\u2010free editing with efficiencies of up to 88% and routine generation of homozygous mutants. By integrating efficient marker\u2010free CRISPR\/Cas9 genome editing and locus\u2010aware expression strategies, we successfully expressed secretory mCherry and human IgG1 Fc fragment at different loci. Our results demonstrate that\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    is a promising expression system for protein production.\n                  <\/jats:p>","DOI":"10.1002\/mlf2.70104","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T04:26:13Z","timestamp":1790742373000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment of a genome editing system and expression of heterologous proteins in the nonconventional yeast\n                    <i>Pichia kudriavzevii<\/i>"],"prefix":"10.1002","author":[{"given":"Jiayin","family":"Ma","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology Chinese Academy of Sciences Beijing China"},{"name":"University of Chinese Academy of Sciences Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifan","family":"Cui","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology Chinese Academy of Sciences Beijing China"},{"name":"University of Chinese Academy of Sciences Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Binru","family":"Yin","sequence":"additional","affiliation":[{"name":"Beijing CAStar Union Sci\u2010Tech Ltd. Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1514-6374","authenticated-orcid":false,"given":"Cheng","family":"Jin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology Chinese Academy of Sciences Beijing China"},{"name":"University of Chinese Academy of Sciences Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"e_1_2_5_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-44997-7"},{"key":"e_1_2_5_3_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13068-020-01729-5"},{"key":"e_1_2_5_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.26745"},{"key":"e_1_2_5_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2013.01.157"},{"key":"e_1_2_5_6_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-025-02159-1"},{"key":"e_1_2_5_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.3c03163"},{"key":"e_1_2_5_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.2c04251"},{"key":"e_1_2_5_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mec.2020.e00124"},{"key":"e_1_2_5_10_1","doi-asserted-by":"publisher","DOI":"10.1128\/mSphere.00345-19"},{"key":"e_1_2_5_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2016.03.027"},{"key":"e_1_2_5_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiosc.2022.07.018"},{"key":"e_1_2_5_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32603-7"},{"key":"e_1_2_5_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/s002940050350"},{"key":"e_1_2_5_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00362084"},{"key":"e_1_2_5_16_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00435290"},{"key":"e_1_2_5_17_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00326538"},{"key":"e_1_2_5_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(85)90187-8"},{"key":"e_1_2_5_19_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00330984"},{"key":"e_1_2_5_20_1","first-page":"4311","article-title":"Modification of Pichia kudriavzevii cells for humanized glycosylation","volume":"50","author":"Ma J","year":"2023","journal-title":"Microbiol China"},{"key":"e_1_2_5_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi800874y"}],"container-title":["mLife"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/mlf2.70104","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/mlf2.70104","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/mlf2.70104","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T04:26:13Z","timestamp":1790742373000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/mlf2.70104"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,29]]},"references-count":20,"alternative-id":["10.1002\/mlf2.70104"],"URL":"https:\/\/doi.org\/10.1002\/mlf2.70104","archive":["Portico"],"relation":{},"ISSN":["2097-1699","2770-100X"],"issn-type":[{"value":"2097-1699","type":"print"},{"value":"2770-100X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,29]]},"assertion":[{"value":"2026-03-04","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-05-29","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-29","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"mlf2.70104"}}