{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T09:57:17Z","timestamp":1791021437876,"version":"4.1.0"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T00:00:00Z","timestamp":1751587200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32460830"],"award-info":[{"award-number":["32460830"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2024YFD2000505"],"award-info":[{"award-number":["2024YFD2000505"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>L-methionine is nutritionally indispensable for humans and animals. It is widely applied to feed, livestock and poultry breeding, food, medicine, energy and chemical industries. Maize endosperm contains a stable protein called <jats:italic>\u03b4-zein<\/jats:italic>, which is abundant in sulfur amino acids, including methionine. <jats:italic>Candida utilis<\/jats:italic> (<jats:italic>C. utilis<\/jats:italic>) has been utilized as a cell factory to express and produce recombinant products. However, there is limited information on its genetic background and expression regulatory elements.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In this study, we aimed to improve methionine yields in an engineered <jats:italic>C. utilis<\/jats:italic> harboring the <jats:italic>\u03b4-zein<\/jats:italic> gene by identifying a strong promoter and optimal signal peptide. A <jats:italic>C. utilis<\/jats:italic> glyceraldehyde-3-phosphate dehydrogenase (<jats:italic>GAP<\/jats:italic>) promoter mutant library was constructed and screened to obtain a strong promoter. Subsequently, <jats:italic>de novo<\/jats:italic> sequencing of the <jats:italic>C. utilis<\/jats:italic> genome was performed using a combination of second-generation Illumina-Seq sequencing platform and third-generation nanopore sequencing technique. Endogenous signal peptides of <jats:italic>C. utilis<\/jats:italic> were analyzed by sequencing the <jats:italic>C. utilis<\/jats:italic> genome. Recombinant <jats:italic>C. utilis<\/jats:italic> strains with homologous integration expression vectors of different signal peptides were constructed and screened for <jats:italic>C. utilis<\/jats:italic> optimal signal peptides for secretion of <jats:italic>\u03b4-zein<\/jats:italic>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Finally, a secretory expression system pGS-zein containing a strong promoter GP6 and an optimal signal peptide SP8 was constructed. In the food-grade engineered <jats:italic>C. utilis<\/jats:italic> C\/pGS-zein methionine content increased by 21.09% compared with that of C\/psP with the original promoter, and by 33.64% compared to wild-type <jats:italic>C. utilis<\/jats:italic>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>This study demonstrates successful expression and secretion of <jats:italic>\u03b4-zein<\/jats:italic> in <jats:italic>C. utilis<\/jats:italic> and establishes a foundation for enhanced methionine production of heterologous proteins in <jats:italic>C. utilis<\/jats:italic>. More importantly, these high-performance biological elements provide fundamental knowledge and technical knowhow for enhanced production of heterologous proteins in <jats:italic>C. utilis<\/jats:italic>.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2025.1586229","type":"journal-article","created":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T05:28:08Z","timestamp":1751606888000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["An optimised promoter and signal peptide improves methionine production of a genetically engineered Candida utilis harboring the \u03b4-zein gene"],"prefix":"10.3389","volume":"16","author":[{"given":"Qiburi","family":"He","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaofeng","family":"Su","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riqilang","family":"Ao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingli","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mei","family":"Chun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gaowa","family":"Gong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,7,4]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"420","DOI":"10.1038\/s41587-019-0036-z","article-title":"SignalP 5.0 improves signal peptide predictions using deep neural networks","volume":"37","author":"Almagro Armenteros","year":"2019","journal-title":"Nat. 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