AOX1 promoter; formate; oxygen transfer rate; sorbitol uptake; Recombinant Proteins
Abstract :
[en] In Komagataella phaffii, the use of formate as an AOX1 promoter (PAOX1) inducer in combination with sorbitol, a non-repressive carbon source, has emerged as a promising alternative to methanol-based expression systems. Recently, we demonstrated that formate derived from the tetrahydrofolate-mediated one-carbon (THF-C1) metabolism accumulates in K. phaffii cells deficient in formate dehydrogenase (FdhKO) when grown in sorbitol-based methanol-free medium. Using the lipase CalB from Candida antarctica as a model protein, we observed that recombinant protein (rProt) productivity in an FdhKO strain grown on sorbitol was comparable to that of an Fdh-proficient strain grown on methanol. However, sorbitol is inefficiently metabolised in K. phaffii, leading to a low growth rate and potentially limiting rProt productivity due to insufficient energy and carbon supply. Here, we increased the sorbitol uptake rate, and thus improved sorbitol metabolism, by overexpressing the gene encoding sorbitol dehydrogenase (SOR1) in an FdhKO strain. Our results demonstrate that while increased sorbitol metabolism promotes biomass formation, it reduces PAOX1 induction, as evidenced by lower formate accumulation and decreased rProt productivity, both for intracellular eGFP and secreted proteins namely CalB lipase and glucose oxidase (Gox) from Aspergillus niger in SOR1-overexpressing strains. Additionally, oxygen availability for cells influences these dynamics, with lower oxygen transfer favouring higher PAOX1 induction due to increased formate accumulation in an FdhKO strain. Our data also suggest that at low oxygen transfer and low sorbitol uptake rate, the proportion of cells in an induced state increased significantly. This work provides valuable insights into the interplay between sorbitol metabolism and oxygen transfer conditions, contributing to the development of improved recombinant protein production strategies in K. phaffii.
Disciplines :
Biotechnology
Author, co-author :
Bustos, Cristina ✱; Microbial Processes and Interactions, TERRA Teaching and Research Centre, Gembloux Agro-Bio Tech, University of Liege, Gembloux, Belgium ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaiso, Chile
Cozmar Quezada, Rocio Celeste ✱; Université de Liège - ULiège > TERRA Research Centre ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaiso, Chile
Berrios, Julio ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaiso, Chile
Fickers, Patrick ; Université de Liège - ULiège > Département GxABT > Microbial technologies
✱ These authors have contributed equally to this work.
Language :
English
Title :
Sorbitol Uptake and Oxygen Transfer Shape AOX1 Promoter Induction in Formate Dehydrogenase-Deficient Komagataella phaffii.
Erasmus+ Erasmus+ Wallonie-Bruxelles International Wallonie-Bruxelles International Wallonie-Bruxelles International Agencia Nacional de Investigación y Desarrollo Agencia Nacional de Investigación y Desarrollo Fondo Nacional de Desarrollo Científico y Tecnológico
Funding text :
Funding: This research was funded by Becas Doctorado Nacional grant number 21211138 and 21211350 Agencia Nacional de Investigaci\u00F3n y Desarrollo (ANID), Chile; Doctoral Internship Scholarship (PUCV, Chile); Research Stay Scholarship (Direcci\u00F3n de Postgrado y Programas, UTFSM, Chile); ERASMUS+ 2022\u20132023 Grant agreement for mobility participants\u2014Higher education (PIC:999854952, E10208749); Wallonie-Bruxelles International through the Cooperation bilateral Belgique-Chilli project SUB/2019/435787 (RIO4), SUB/2023/591923/MOD (RI06) and SUB/2023/585456 (RC06). FONDECYT Regular (project number 1191196), University of Liege, Terra Teaching and Research Center. The authors thank L. Henrion, V. Vandenbroucke, A. Padmanabhan, Korka, S. Telek, A. Zicler, S. Steels, and R. Thomas for their technical assistance and valuable discussions. The graphical abstract was created with BioRender.com. This research was funded by Becas Doctorado Nacional grant number 21211138 and 21211350 Agencia Nacional de Investigaci\u00F3n y Desarrollo (ANID), Chile; Doctoral Internship Scholarship (PUCV, Chile); Research Stay Scholarship (Direcci\u00F3n de Postgrado y Programas, UTFSM, Chile); ERASMUS+ 2022\u20132023 Grant agreement for mobility participants\u2014Higher education (PIC:999854952, E10208749); Wallonie-Bruxelles International through the Cooperation bilateral Belgique-Chilli project SUB/2019/435787 (RIO4), SUB/2023/591923/MOD (RI06) and SUB/2023/585456 (RC06). FONDECYT Regular (project number 1191196), University of Liege, Terra Teaching and Research Center.The authors thank L. Henrion, V. Vandenbroucke, A. Padmanabhan, Korka, S. Telek, A. Zicler, S. Steels, and R. Thomas for their technical assistance and valuable discussions. The graphical abstract was created with BioRender.com . This research was funded by Becas Doctorado Nacional grant number 21211138 and 21211350 Agencia Nacional de Investigaci\u00F3n y Desarrollo (ANID), Chile; Doctoral Internship Scholarship (PUCV, Chile); Research Stay Scholarship (Direcci\u00F3n de Postgrado y Programas, UTFSM, Chile); ERASMUS+ 2022\u20132023 Grant agreement for mobility participants\u2014Higher education (PIC:999854952, E10208749); Wallonie\u2010Bruxelles International through the Cooperation bilateral Belgique\u2010Chilli project SUB/2019/435787 (RIO4), SUB/2023/591923/MOD (RI06) and SUB/2023/585456 (RC06). FONDECYT Regular (project number 1191196), University of Liege, Terra Teaching and Research Center.
Akentyev, P., D. Sokolova, A. Korzhenkov, I. Gubaidullin, and D. Kozlov. 2023. “Expression Level of SOR1 Is a Bottleneck for Efficient Sorbitol Utilization by Yeast Komagataella Kurtzmanii.” Yeast 40: 414–424.
Berrios, J., C. W. Theron, S. Steels, et al. 2022. “Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism.” Microorganisms 10: 1466.
Bustos, C., J. Berrios, and P. Fickers. 2024. “Formate From THF-C1 Metabolism Induces the AOX1 Promoter in Formate Dehydrogenase-Deficient Komagataella phaffii.” Microbial Biotechnology 17: e70022.
Carly, F., H. Niu, F. Delvigne, and P. Fickers. 2016. “Influence of Methanol/Sorbitol Co-Feeding Rate on pAOX1 Induction in a Pichia pastoris Mut+ Strain in Bioreactor With Limited Oxygen Transfer Rate.” Journal of Industrial Microbiology & Biotechnology 43: 517–523.
Carnicer, M., K. Baumann, I. Töplitz, et al. 2009. “Macromolecular and Elemental Composition Analysis and Extracellular Metabolite Balances of Pichia pastoris Growing at Different Oxygen Levels.” Microbial Cell Factories 8: 1–14.
Çelik, E., P. Çalık, and S. G. Oliver. 2009. “Fed-Batch Methanol Feeding Strategy for Recombinant Protein Production by Pichia pastoris in the Presence of Co-Substrate Sorbitol.” Yeast 26: 473–484.
Christensen, K. E., and R. E. Mackenzie. 2006. “Mitochondrial One-Carbon Metabolism Is Adapted to the Specific Needs of Yeast, Plants and Mammals.” BioEssays 28: 595–605.
Ergün, B. G., J. Berrios, B. Binay, and P. Fickers. 2021. “Recombinant Protein Production in Pichia pastoris: From Transcriptionally Redesigned Strains to Bioprocess Optimization and Metabolic Modelling.” FEMS Yeast Research 21: foab057.
Ergün, B. G., K. Laçın, B. Çaloğlu, and B. Binay. 2022. “Second Generation Pichia pastoris Strain and Bioprocess Designs.” Biotechnology for Biofuels and Bioproducts 15: 150.
Feng, A., J. Zhou, H. Mao, H. Zhou, and J. Zhang. 2022. “Heterologous Protein Expression Enhancement of Komagataella phaffii by Ammonium Formate Induction Based on Transcriptomic Analysis.” Biochemical Engineering Journal 185: 108503.
Fickers, P., J. M. Nicaud, J. Destain, and P. Thonart. 2003. “Overproduction of Lipase by Yarrowia lipolytica Mutants.” Applied Microbiology and Biotechnology 63: 136–142.
Gorczyca, M., J. Kaźmierczak, S. Steels, P. Fickers, and E. Celińska. 2020. “Impact of Oxygen Availability on Heterologous Gene Expression and Polypeptide Secretion Dynamics in Yarrowia lipolytica-Based Protein Production Platforms.” Yeast 37: 559–568.
Hartner, F. S., and A. Glieder. 2006. “Regulation of Methanol Utilisation Pathway Genes in Yeasts.” Microbial Cell Factories 5: 39.
Jayachandran, C., B. Palanisamy Athiyaman, and M. Sankaranarayanan. 2017. “Formate Co-Feeding Improved Candida antarctica Lipase B Activity in Pichia pastoris.” Research Journal of Biotechnology 12: 29–36.
Jordan, P., J. Y. Choe, E. Boles, and M. Oreb. 2016. “Hxt13, Hxt15, Hxt16 and Hxt17 From Saccharomyces cerevisiae Represent a Novel Type of Polyol Transporters.” Scientific Reports 6: 23502.
Liu, B., H. Li, H. Zhou, and J. Zhang. 2022. “Enhancing Xylanase Expression by Komagataella phafi by Formate as Carbon Source and Inducer.” Applied Microbiology and Biotechnology 106: 7819–7829.
Lv, X., W. Yu, C. Zhang, et al. 2023. “C1-Based Biomanufacturing: Advances, Challenges and Perspestives.” Bioresource Technology 367: 128259.
Niu, H., L. Jost, N. Pirlot, et al. 2013. “A Quantitative Study of Methanol/Sorbitol Co-Feeding Process of a Pichia pastoris Mut+/pAOX1-lacZ Strain.” Microbial Cell Factories 12: 33.
Prielhofer, R., J. J. Barrero, S. Steuer, et al. 2017. “GoldenPiCS: A Golden Gate-Derived Modular Cloning System for Applied Synthetic Biology in the Yeast Pichia pastoris.” BMC Systems Biology 11: 123.
Sassi, H., F. Delvigne, T. Kar, et al. 2016. “Deciphering How LIP2 and POX2 Promoters Can Optimally Regulate Recombinant Protein Production in the Yeast Yarrowia lipolytica.” Microbial Cell Factories 15: 159.
Singh, A., and A. Narang. 2020. “The Mut+ Strain of Komagataella phaffii (Pichia pastoris) Expresses pAOX1 5 and 10 Times Faster Than Muts and Mut− Strains: Evidence That Formaldehyde or/and Formate Are True Inducers of pAOX1.” Applied Microbiology and Biotechnology 104: 7801–7814.
Singh, A., and A. Narang. 2023. “PAOX1 Expression in Mixed-Substrate Continuous Cultures of Komagataella phaffii (Pichia pastoris) is Completely Determined by Methanol Consumption Regardless of the Secondary Carbon Source.” Frontiers in Bioengineering and Biotechnology 11: 1123703.
Theron, C. W., J. Berrios, S. Steels, et al. 2019. “Expression of Recombinant Enhanced Green Fluorescent Protein Provides Insight Into Foreign Gene-Expression Differences Between Mut+ and MutS Strains of Pichia pastoris.” Yeast 36: 285–296.
Toivari, M. H., L. Salusjärvi, L. Ruohonen, and M. Penttilä. 2004. “Endogenous Xylose Pathway in Saccharomyces cerevisiae.” Applied and Environmental Microbiology 70: 3681–3686.
Tyurin, O. V., and D. G. Kozlov. 2015. “Deletion of the FLD Gene in Methylotrophic Yeasts Komagataella phaffii and Komagataella kurtzmanii Results in Enhanced Induction of the AOX1 Promoter in Response to Either Methanol or Formate.” Microbiology (N Y) 84: 408–411.
Velastegui, E., J. Quezada, C. Altamirano, J. Berrios, and P. Fickers. 2023. “Co-Feeding Strategy Alleviates Hypoxic Stress in Large-Scale Bioreactor for Optimal Production of Secretory Recombinant Proteins in Pichia pastoris.” Journal of Biotechnology 373: 20–23.
Velastegui, E., C. Theron, J. Berrios, and P. Fickers. 2019. “Downregulation by Organic Nitrogen of AOX1 Promoter Used for Controlled Expression of Foreign Genes in the Yeast Pichia pastoris.” Yeast 36: 297–304.
