{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T11:03:12Z","timestamp":1790593392095,"version":"4.1.0"},"reference-count":46,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T00:00:00Z","timestamp":1789430400000},"content-version":"vor","delay-in-days":14,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["enviromicro-journals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Microbial Biotechnology"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Pichia kudriavzevii<\/jats:italic>\n                    is gaining attention as a non\u2010conventional yeast with significant industrial potential, owing to its robust tolerance to low pH and diverse process\u2010related stresses. However, the lack of a reliable episomal expression system and the scarcity of well\u2010characterized promoters currently impede predictable gene expression and modular metabolic engineering in this workhorse. Here, we report development of the stable episomal expression vector for\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    E1. This was achieved through identification of a functional autonomously replicating sequence from the endogenous genome, guided by the established host\u2010vector GC compatibility workflow. Leveraging this episomal platform, we then constructed an endogenous promoter library for\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    E1 via transcriptomic sequencing data analysis. Among the characterized promoters, the endogenous PkTDH3 promoter exhibited the highest activity. Notably, PkTDH3 demonstrated more stable and sustained expression compared to the strong promoter ScTDH3 from\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                    <\/jats:styled-content>\n                    , underscoring the value of establishing native promoter resources for\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    . Furthermore, we expanded the strength range of the promoter library by engineering the upstream activating sequence of PkTDH3 through random mutagenesis. As a proof of concept, the developed promoter library was employed to construct\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    mutants with elevated protein content, demonstrating the utility of this integrated genetic toolkit for strain engineering in\n                    <jats:italic>P. kudriavzevii<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1111\/1751-7915.70447","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:43:36Z","timestamp":1789537416000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Expanding the Genetic Toolkit of\n                    <i>Pichia kudriavzevii<\/i>\n                    <scp>E1<\/scp>\n                    for Enhanced Mycoprotein Production"],"prefix":"10.1111","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5189-7917","authenticated-orcid":false,"given":"Yupeng","family":"Wang","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"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4280-4915","authenticated-orcid":false,"given":"Shenghan","family":"Gao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology, Chinese Academy of Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-0608-9963","authenticated-orcid":false,"given":"Chenchen","family":"Wu","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"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-9404-4449","authenticated-orcid":false,"given":"Liangchen","family":"Guo","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"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8342-739X","authenticated-orcid":false,"given":"Peijie","family":"Han","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology, Chinese Academy of Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9916-5232","authenticated-orcid":false,"given":"Limin","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology, Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Key Laboratory of Genetic Element Biosourcing &amp; Intelligent Design for Biomanufacturing Institute of Microbiology, Chinese Academy of Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2850-6073","authenticated-orcid":false,"given":"Justyna","family":"Rucha\u0142a","sequence":"additional","affiliation":[{"name":"Faculty of Biotechnology, Medical College University of Rzesz\u00f3w  Rzesz\u00f3w Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3148-0986","authenticated-orcid":false,"given":"Bo","family":"Yu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization Institute of Microbiology, Chinese Academy of Sciences  Beijing China"},{"name":"Beijing Key Laboratory of Genetic Element Biosourcing &amp; Intelligent Design for Biomanufacturing Institute of Microbiology, Chinese Academy of Sciences  Beijing 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