{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,14]],"date-time":"2026-06-14T12:52:59Z","timestamp":1781441579323,"version":"3.54.1"},"reference-count":0,"publisher":"Knowledge E DMCC","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJM"],"abstract":"<jats:p>Background and Objectives: This study aimed to enhance glucose oxidase (GOX) production in Pichia pastoris GS115 us- ing a novel dual-promoter system, combining the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) in pGAPZ\u03b1A with the methanol-inducible Alcohol oxidase 1 promoter (pAOX1) in PIC9.\r\nMaterials and Methods: The GOX gene from Aspergillus niger (ATCC 9029) and a transcription factor, general control nonderepressible 4 (GCN4) gene from P. pastoris were co-expressed to mitigate oxidative stress, thereby improving cell viability and enzyme yield. The recombinant construct pGAPZ\u03b1A-GOX-GCN4 was transformed into P. pastoris GS115 and P. pastoris GS115-PIC9 via electroporation. Expression conditions under various temperatures and pH treatments were optimized. We examined glucose oxidase expression by inducing methanol at concentrations of 100% and 5% in BMMY (Buffered Methanol-complex-medium). The highest enzyme levels were observed at pH 6.0, 34\u00b0C, and 5% methanol induc- tion. Enzyme validation was performed using SDS and Western blotting.\r\nResults: Co-expression of GCN4 significantly enhanced GOX production, achieving 16.65 \u00b5g\/mL (333 U\/mL) in P. pastoris GS115-PIC9-pGAPZ\u03b1A-GOX-GCN4(2), a 377.4-fold increase over the control, and 11.03 \u00b5g\/mL (220.6 U\/mL) in P. pasto- ris GS115-PIC9-pGAPZ\u03b1A-GOX-GCN4(3), a 249.65-fold increase.\r\nConclusion: The results demonstrate that GCN4's stress mitigation amplifies the synergy between constitutive and inducible promoters. The dual-promoter strategy offers a robust platform for recombinant protein production.<\/jats:p>","DOI":"10.18502\/ijm.v18i3.21670","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T11:48:09Z","timestamp":1780487289000},"source":"Crossref","is-referenced-by-count":0,"title":["Simultaneous GCN4 co-expression and promoter optimization enhance glucose oxidase production in Pichia pastoris"],"prefix":"10.18502","author":[{"given":"Nasrin","family":"Ghafari","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammad Reza","family":"Zolfaghari","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamshid","family":"Raheb","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali Asghar","family":"Karkhane","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seyed Soheil","family":"Aghaei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bijan","family":"Bambai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amena","family":"Mohaghegh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"7770","published-online":{"date-parts":[[2026,6,3]]},"container-title":["Iranian Journal of Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/publish.kne-publishing.com\/index.php\/IJM\/article\/download\/21670\/20151","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/publish.kne-publishing.com\/index.php\/IJM\/article\/download\/21670\/20151","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,14]],"date-time":"2026-06-14T12:12:01Z","timestamp":1781439121000},"score":1,"resource":{"primary":{"URL":"https:\/\/publish.kne-publishing.com\/index.php\/IJM\/article\/view\/21670"},"secondary":[{"URL":"https:\/\/ijm.tums.ac.ir\/index.php\/ijm\/article\/view\/5623","label":"UNIVERSITY"}]},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,3]]},"references-count":0,"URL":"https:\/\/doi.org\/10.18502\/ijm.v18i3.21670","relation":{},"ISSN":["2008-4447","2008-3289"],"issn-type":[{"value":"2008-4447","type":"electronic"},{"value":"2008-3289","type":"print"}],"subject":[],"published":{"date-parts":[[2026,6,3]]}}}