{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,16]],"date-time":"2026-08-16T07:31:35Z","timestamp":1786865495823,"version":"build-2736575974"},"reference-count":61,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T00:00:00Z","timestamp":1776470400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100017170","name":"Thailand Science Research and Innovation","doi-asserted-by":"publisher","award":["FFB680075\/0337"],"award-info":[{"award-number":["FFB680075\/0337"]}],"id":[{"id":"10.13039\/501100017170","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017170","name":"Thailand Science Research and Innovation","doi-asserted-by":"publisher","award":["FFB690024\/0337"],"award-info":[{"award-number":["FFB690024\/0337"]}],"id":[{"id":"10.13039\/501100017170","id-type":"DOI","asserted-by":"publisher"}]},{"award":["FFB680075\/0337"],"award-info":[{"award-number":["FFB680075\/0337"]}],"id":[{"id":"https:\/\/ror.org\/000025p04","id-type":"ROR","asserted-by":"publisher"}]},{"award":["FFB690024\/0337"],"award-info":[{"award-number":["FFB690024\/0337"]}],"id":[{"id":"https:\/\/ror.org\/000025p04","id-type":"ROR","asserted-by":"publisher"}]},{"name":"the National Science, Research and Innovation Fund"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae, a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae. We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1, a Sec1\/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor\u2013mediated vesicle trafficking, which, in combination with SPAoalp1, increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine \u03ba-casein increased secretion from 0.11 to 0.24 mg\/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC90 values of 4.56\u20138.24% (v\/v) against two Candida albicans strains and 4.68% (v\/v) against Aspergillus niger. The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production.<\/jats:p>","DOI":"10.3390\/jof12040289","type":"journal-article","created":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T08:24:23Z","timestamp":1776673463000},"page":"289","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Two-Step Engineering of Food-Grade Aspergillus oryzae via Endogenous Signal Peptides and Vesicle Trafficking Proteins to Enhance Carrier-Free Protein Secretion"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6482-8750","authenticated-orcid":false,"given":"Sarocha","family":"Panchanawaporn","sequence":"first","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nakul","family":"Rattanaphan","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2559-0014","authenticated-orcid":false,"given":"Sukanya","family":"Jeennor","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1978-2535","authenticated-orcid":false,"given":"Jutamas","family":"Anantayanon","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9715-0376","authenticated-orcid":false,"given":"Weerapong","family":"Woraprayote","sequence":"additional","affiliation":[{"name":"Food Biotechnology Research Team, Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4640-4273","authenticated-orcid":false,"given":"Laphaslada","family":"Pumpuang","sequence":"additional","affiliation":[{"name":"Food Biotechnology Research Team, Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thipphiya","family":"Karirat","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-9709-8011","authenticated-orcid":false,"given":"Nuttamon","family":"Prompakdee","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7263-4892","authenticated-orcid":false,"given":"Kobkul","family":"Laoteng","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0657-2897","authenticated-orcid":false,"given":"Chanikul","family":"Chutrakul","sequence":"additional","affiliation":[{"name":"Industrial Bioprocess Technology Research Team (IIBT), Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Nguyen, T., Karl, M., and Santini, A. 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