Iranian journal of microbiology · 2026
Simultaneous GCN4 co-expression and promoter optimization enhance glucose oxidase production in Pichia pastoris.
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Ghafari Nasrin, Zolfaghari Mohammad Reza, Raheb Jamshid, Karkhane Ali Asghar, Aghaei Seyed Soheil, Bambai Bijan, Mohaghegh Amena
Аннотация
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.
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.