Simultaneous GCN4 co-expression and promoter optimization enhance glucose oxidase production in Pichia pastoris
-
Nasrin Ghafari
,
Mohammad Reza Zolfaghari
,
Jamshid Raheb
,
Ali Asghar Karkhane
,
Seyed Soheil Aghaei
,
Bijan Bambai
,
Amena Mohaghegh
Abstract
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.
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5. Ayenimo JG, Adeloju SB. Inhibitive potentiometric detection of trace metals with ultrathin polypyrrole glucose oxidase biosensor. Talanta 2015; 137: 62-70.
6. Macauley‐Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005; 22: 249-270.
7. Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, et al. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 2009; 8: 29.
8. Abad S, Nahalka J, Winkler M, Bergler G, Speight R, Glieder A, et al. High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris. Biotechnol Lett 2011; 33: 557-563.
9. Duman-Özdamar ZE, Binay B. Production of industrial enzymes via Pichia pastoris as a cell factory in bioreactor: current status and future aspects. Protein J 2021; 40: 367-376.
10. Zha J, Liu D, Ren J, Liu Z, Wu X. Advances in metabolic engineering of Pichia pastoris strains as powerful cell factories. J Fungi (Basel) 2023; 9: 1027.
11. Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D. Metabolic engineering of Pichia pastoris. Metab Eng 2018; 50: 2-15.
12. Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 2002; 13: 329-332.
13. Heyland J, Fu J, Blank LM. Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production. Biotechnol Bioeng 2010; 107: 357-368.
14. Lee CC, Williams TG, Wong DWS, Robertson GH. An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid 2005; 54: 80-85.
15. De Schutter K, Lin YC, Tiels P, Van Hecka A, Glinka S, Weber-Lehmann J, et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 2009; 27: 561-566.
16. Shen W, Xue Y, Liu Y, Kong C, Wang X, Huang M, et al. A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb Cell Fact 2016; 15: 178.
17. Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 2005; 18: 119-138.
18. Nevoigt E, Fischer C, Mucha O, Matthaus F, Stahl U, Stephanopulos G. Engineering promoter regulation. Biotechnol Bioeng 2007; 96: 550-558.
19. Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. Expression in the yeast Pichia pastoris. Methods Enzymol 2009; 463: 169-189.
20. Gasser B, Steiger MG, Mattanovich D. Methanol regulated yeast promoters: production vehicles and toolbox for synthetic biology. Microb Cell Fact 2015; 14: 196.
21. Yu XW, Lu X, Zhao LS, Xu Y. Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris. Process Biochem 2013; 48: 1462-1468.
22. Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smit LP, et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997; 190: 55-62.
23. Stils HF. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR J 2005; 46: 280-293.
24. Waterham HR, Digan ME, Koutz PK, Lair AV, Cregg JM. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 1997; 186: 37-44.
25. Stadlmayr G, Mecklenbräuker A, Rothmuller M, Maurer M, Sauer M, Mattanovich D, et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 2010; 150: 519-529.
26. Qiu Z, Guo Y, Bao X, Hao J, Sun G, Peng B, et al. Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168. Biotechnol Biotechnol Equip 2016; 30: 998-1005.
27. Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY, et al. Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep 2009; 36: 1611-1619.
28. Wu JM, Chieng LL, Hsu TA, Lee CK. Sequential expression of recombinant proteins and their separate recovery from a Pichia pastoris cultivation. Biochem Eng J 2003; 16: 9-16.
29. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26: 509-515.
30. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al . An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 2003; 11: 619-633.
31. Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 2010; 9: 49.
32. Deprez RHL, Fijnvandraat AC, Ruijter JM, Moorman AFM. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 2002; 307: 63-69.
33. Petruccioli M, Piccioni P, Federici F, Polsinelli. Glucose oxidase overproducing mutants of Penicillium variabile (P16). FEMS Microbiol Lett 1995; 128: 107-111.
34. Du C, Han L, Xiao A, Cao M. Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris. J Microbiol Biotechnol 2011; 21: 1264-1269.
35. Evans JK, Troilo P, Ledwith BJ. Simultaneous purification of RNA and DNA from liver using sodium acetate precipitation. Biotechniques 1998; 24: 416-418.
36. Kapat A, Jung JK, Park YH. Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition. J Appl Microbiol 2001; 90: 216-222.
37. Freier L, Hemmerich J, Schöler K, Wiechert W, Oldiges M, von Lieres E, et al. Framework for Kriging‐based iterative experimental analysis and design: Optimization of secretory protein production in Corynebacterium glutamicum. Eng Life Sci 2016; 16: 538-549.
38. Garcia-Ortega X, Ferrer P, Montesinos JL, Valero F. Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter. Biochem Eng J 2013; 79: 172-181.
39. Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S-O. Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 2005; 27: 399-406.
40. Gonçalves AM, Pedro AQ, Maia C, Sousa F, Queiroz JA, Passarinha LA. Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins. J Microbiol Biotechnol 2013; 23: 587-601.
41. Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D. Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 2008; 100: 177-183.
42. Várnai A, Tang C, Bengtsson O, Atterton A, Mathiesen G, Eijsink VG. Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Microb Cell Fact 2014; 13: 57.
43. Shi XZ, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D. Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 2003; 28: 321-330.
44. Jiménez-Martí E, Zuzuarregui A, Gomar-Alba M, Gutiérrez D, Gil C, Del Olmo M. Molecular response of Saccharomyces cerevisiae wine and laboratory strains to high sugar stress conditions. Int J Food Microbiol 2011; 145: 211-220.
45. Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P. Scaling-up fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci Rep 2016; 6: 18439.
46. Yu Y, Zhou X, Wu S, Wei T, Yu L. High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation. Electron. J Biotechnol 2014; 17: 311-316.
47. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, et al. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008; 7: 11.
48. Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact 2012; 11: 22.
49. Ramón R, Ferrer P, Valero F. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 2007; 130: 39-46.
50. Inan M, Meagher MM. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 2001; 92: 585-589.
51. Landes N, Gasser B, Vorauer-Uhl K, Lhota G, Mattanovich D, Maurer M. The vitamin‐sensitive promoter PTHI11 enables pre‐defined autonomous induction of recombinant protein production in Pichia pastoris. Biotechnol Bioeng 2016; 113: 2633-2643.
52. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002; 30: 503-512.
53. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F. Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol 2005; 116: 321-335.
54. Çelik E, Çalık P, Oliver SG. Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: effects of methanol feeding rate. Biotechnol Bioeng 2010; 105: 317-329.
2. Petruccioli M, Federici F, Bucke C, Keshavarz T. Enhancement of glucose oxidase production by Penicillium variabile P16. Enzyme Microb Technol 1999; 24:397-401.
3. Zeeb B, Fischer L, Weiss J. Stabilization of food dispersions by enzymes. Food Funct 2014; 5: 198-213.
4. Taboada-Puig R, Junghanns C, Demarche P, Moreira M, Feijoo G, Lema J, et al. Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: Production, partial characterization and application for the elimination of endocrine disruptors. Bioresour Technol 2011; 102: 6593-6599.
5. Ayenimo JG, Adeloju SB. Inhibitive potentiometric detection of trace metals with ultrathin polypyrrole glucose oxidase biosensor. Talanta 2015; 137: 62-70.
6. Macauley‐Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005; 22: 249-270.
7. Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, et al. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 2009; 8: 29.
8. Abad S, Nahalka J, Winkler M, Bergler G, Speight R, Glieder A, et al. High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris. Biotechnol Lett 2011; 33: 557-563.
9. Duman-Özdamar ZE, Binay B. Production of industrial enzymes via Pichia pastoris as a cell factory in bioreactor: current status and future aspects. Protein J 2021; 40: 367-376.
10. Zha J, Liu D, Ren J, Liu Z, Wu X. Advances in metabolic engineering of Pichia pastoris strains as powerful cell factories. J Fungi (Basel) 2023; 9: 1027.
11. Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D. Metabolic engineering of Pichia pastoris. Metab Eng 2018; 50: 2-15.
12. Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 2002; 13: 329-332.
13. Heyland J, Fu J, Blank LM. Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production. Biotechnol Bioeng 2010; 107: 357-368.
14. Lee CC, Williams TG, Wong DWS, Robertson GH. An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid 2005; 54: 80-85.
15. De Schutter K, Lin YC, Tiels P, Van Hecka A, Glinka S, Weber-Lehmann J, et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 2009; 27: 561-566.
16. Shen W, Xue Y, Liu Y, Kong C, Wang X, Huang M, et al. A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb Cell Fact 2016; 15: 178.
17. Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 2005; 18: 119-138.
18. Nevoigt E, Fischer C, Mucha O, Matthaus F, Stahl U, Stephanopulos G. Engineering promoter regulation. Biotechnol Bioeng 2007; 96: 550-558.
19. Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. Expression in the yeast Pichia pastoris. Methods Enzymol 2009; 463: 169-189.
20. Gasser B, Steiger MG, Mattanovich D. Methanol regulated yeast promoters: production vehicles and toolbox for synthetic biology. Microb Cell Fact 2015; 14: 196.
21. Yu XW, Lu X, Zhao LS, Xu Y. Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris. Process Biochem 2013; 48: 1462-1468.
22. Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smit LP, et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997; 190: 55-62.
23. Stils HF. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR J 2005; 46: 280-293.
24. Waterham HR, Digan ME, Koutz PK, Lair AV, Cregg JM. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 1997; 186: 37-44.
25. Stadlmayr G, Mecklenbräuker A, Rothmuller M, Maurer M, Sauer M, Mattanovich D, et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 2010; 150: 519-529.
26. Qiu Z, Guo Y, Bao X, Hao J, Sun G, Peng B, et al. Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168. Biotechnol Biotechnol Equip 2016; 30: 998-1005.
27. Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY, et al. Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep 2009; 36: 1611-1619.
28. Wu JM, Chieng LL, Hsu TA, Lee CK. Sequential expression of recombinant proteins and their separate recovery from a Pichia pastoris cultivation. Biochem Eng J 2003; 16: 9-16.
29. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004; 26: 509-515.
30. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al . An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 2003; 11: 619-633.
31. Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, et al. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 2010; 9: 49.
32. Deprez RHL, Fijnvandraat AC, Ruijter JM, Moorman AFM. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 2002; 307: 63-69.
33. Petruccioli M, Piccioni P, Federici F, Polsinelli. Glucose oxidase overproducing mutants of Penicillium variabile (P16). FEMS Microbiol Lett 1995; 128: 107-111.
34. Du C, Han L, Xiao A, Cao M. Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris. J Microbiol Biotechnol 2011; 21: 1264-1269.
35. Evans JK, Troilo P, Ledwith BJ. Simultaneous purification of RNA and DNA from liver using sodium acetate precipitation. Biotechniques 1998; 24: 416-418.
36. Kapat A, Jung JK, Park YH. Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition. J Appl Microbiol 2001; 90: 216-222.
37. Freier L, Hemmerich J, Schöler K, Wiechert W, Oldiges M, von Lieres E, et al. Framework for Kriging‐based iterative experimental analysis and design: Optimization of secretory protein production in Corynebacterium glutamicum. Eng Life Sci 2016; 16: 538-549.
38. Garcia-Ortega X, Ferrer P, Montesinos JL, Valero F. Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter. Biochem Eng J 2013; 79: 172-181.
39. Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S-O. Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 2005; 27: 399-406.
40. Gonçalves AM, Pedro AQ, Maia C, Sousa F, Queiroz JA, Passarinha LA. Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins. J Microbiol Biotechnol 2013; 23: 587-601.
41. Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D. Hypoxic fed‐batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 2008; 100: 177-183.
42. Várnai A, Tang C, Bengtsson O, Atterton A, Mathiesen G, Eijsink VG. Expression of endoglucanases in Pichia pastoris under control of the GAP promoter. Microb Cell Fact 2014; 13: 57.
43. Shi XZ, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D. Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 2003; 28: 321-330.
44. Jiménez-Martí E, Zuzuarregui A, Gomar-Alba M, Gutiérrez D, Gil C, Del Olmo M. Molecular response of Saccharomyces cerevisiae wine and laboratory strains to high sugar stress conditions. Int J Food Microbiol 2011; 145: 211-220.
45. Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P. Scaling-up fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci Rep 2016; 6: 18439.
46. Yu Y, Zhou X, Wu S, Wei T, Yu L. High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation. Electron. J Biotechnol 2014; 17: 311-316.
47. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, et al. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008; 7: 11.
48. Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact 2012; 11: 22.
49. Ramón R, Ferrer P, Valero F. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 2007; 130: 39-46.
50. Inan M, Meagher MM. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 2001; 92: 585-589.
51. Landes N, Gasser B, Vorauer-Uhl K, Lhota G, Mattanovich D, Maurer M. The vitamin‐sensitive promoter PTHI11 enables pre‐defined autonomous induction of recombinant protein production in Pichia pastoris. Biotechnol Bioeng 2016; 113: 2633-2643.
52. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002; 30: 503-512.
53. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F. Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol 2005; 116: 321-335.
54. Çelik E, Çalık P, Oliver SG. Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: effects of methanol feeding rate. Biotechnol Bioeng 2010; 105: 317-329.
| Files | ||
| Issue | Vol 18 No 3 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i3.21670 | |
| Keywords | ||
| Pichia pastoris Glucose oxidase GAP promoter AOX1 promoter GCN4 Dual-promoter expression Recombinant protein Methanol induction Oxidative stress | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Ghafari N, Zolfaghari MR, Raheb J, Karkhane AA, Aghaei SS, Bambai B, Mohaghegh A. Simultaneous GCN4 co-expression and promoter optimization enhance glucose oxidase production in Pichia pastoris. Iran J Microbiol. 2026;18(3):428-438.
