Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 3 2026 06 07 428 438 Nasrin Ghafari Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran; Department of Microbiology, Qo.C, Islamic Azad University, Qom, Iran Mohammad Reza Zolfaghari Department of Microbiology, Qo.C, Islamic Azad University, Qom, Iran Jamshid Raheb Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Ali Asghar Karkhane Department of Bioprocess Engineering, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Seyed Soheil Aghaei Department of Microbiology, Qo.C, Islamic Azad University, Qom, Iran Bijan Bambai Department of Systems Biotechnology, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Amena Mohaghegh Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran 2025 07 13 2025 12 09 Background and Objectives: This study aimed to enhance glucose oxidase (GOX) production in Pichia pastoris GS115 using a novel dual-promoter system, combining the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) in pGAPZαA with the methanol-inducible Alcohol oxidase 1 promoter (pAOX1) in PIC9. Materials 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 pGAPZαA-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°C, and 5% methanol induction. Enzyme validation was performed using SDS and Western blotting. Results: Co-expression of GCN4 significantly enhanced GOX production, achieving 16.65 µg/mL (333 U/mL) in P. pastoris GS115-PIC9-pGAPZαA-GOX-GCN4(2), a 377.4-fold increase over the control, and 11.03 µg/mL (220.6 U/mL) in P. pastoris GS115-PIC9-pGAPZαA-GOX-GCN4(3), a 249.65-fold increase. Conclusion: 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. https://ijm.tums.ac.ir/index.php/ijm/article/view/5623 https://ijm.tums.ac.ir/index.php/ijm/article/download/5623/1903