Bioreactor; Komagataella phaffii; Phenotypic heterogeneity; Pichia pastoris; Recombinant protein; Scale-down; eGFP; Methanol; Recombinant Proteins; Bioreactors; Recombinant Proteins/metabolism; Hypoxia; Methanol/metabolism; Pichia/genetics; Pichia/metabolism; Cell residence time; Culture conditions; Enhanced green fluorescent protein; Komagataellum phaffii; Large scale bioreactors; Medium heterogeneity; Protein synthesis; Pichia; Biotechnology; Applied Microbiology and Biotechnology
Abstract :
[en] Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.
Disciplines :
Microbiology
Author, co-author :
Velastegui, Edgar ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av Brasil 2085, Valparaiso, 2340000, Chile ; Microbial Processes and Interactions, TERRA Teaching and Research Centre, Gembloux Agro Bio Tech, University of Liege, Gembloux, Belgium
Quezada, Johan ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av Brasil 2085, Valparaiso, 2340000, Chile
Guerrero, Karlo ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av Brasil 2085, Valparaiso, 2340000, Chile
Altamirano, Claudia ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av Brasil 2085, Valparaiso, 2340000, Chile
Berrios, Julio ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av Brasil 2085, Valparaiso, 2340000, Chile. julio.berrios@pucv.cl
Fickers, Patrick ; Université de Liège - ULiège > TERRA Research Centre > Microbial technologies
Language :
English
Title :
Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?
Publication date :
April 2023
Journal title :
Applied Microbiology and Biotechnology
ISSN :
0175-7598
eISSN :
1432-0614
Publisher :
Springer Science and Business Media Deutschland GmbH, Germany
FONDECYT - National Fund for Scientific and Technological Development [CL] ANID - Agencia Nacional de Investigación y Desarrollo [CL] WBI - Wallonie-Bruxelles International [BE]
Funding text :
This research was funded by the FONDECYT Regular (project 1191196), the ANILLO Regular de Ciencia y Tecnología (project ACT210068), the Fondecyt Postdoctorado (project 3220826) and Beca Doctorado Nacional (N° 21170349 and 21191422) from the Agencia Nacional de Investigación y Desarrollo (ANID), Chile; and the Wallonie-Bruxelles International through the Cooperation bilateral Belgique-Chili project SUB/2019/435787.
Adelantado N, Tarazona P, Grillitsch K, García-Ortega X, Monforte S, Valero F, Feussner I, Daum G, Ferrer P (2017) The effect of hypoxia on the lipidome of recombinant Pichia pastoris. Microb Cell Fact 16:1–15. 10.1186/s12934-017-0699-4 DOI: 10.1186/s12934-017-0699-4
Ata O, Gunduz B, Fickers P, Heistinger L, Mattanovich D, Rebnegger C, Gasser B (2021) What makes Komagataella phaffii non-conventional ? FEMS Yeast Res 21:1–15. 10.1093/femsyr/foab059 DOI: 10.1093/femsyr/foab059
Baumann K, Carnicer M, Dragosits M, Graf A, Stadlmann J, Jouhten P, Maaheimo H, Gasser B, Albiol J, Mattanovich D, Ferrer P (2010) A multi-level study of recombinant Pichia pastoris in different oxygen conditions. BMC Syst Biol 4:1–22. 10.1186/1752-0509-4-141 DOI: 10.1186/1752-0509-4-141
Berrios J, Flores M, Díaz-Barrera A, Altamirano C, Martínez I, Cabrera Z (2017) A comparative study of glycerol and sorbitol as co-substrates in methanol-induced cultures of Pichia pastoris: temperature effect and scale-up simulation. J Ind Microbiol Biotechnol 44:407–411. 10.1007/s10295-016-1895-7 DOI: 10.1007/s10295-016-1895-7
Berrios J, Theron CW, Steels S, Ponce B, Velastegui E, Bustos C, Altamirano C, Fickers P (2022) Role of dissimilative pathway of Komagataella phaffii (Pichia pastoris): formaldehyde toxicity and energy metabolism. Microorganisms 10. 10.3390/microorganisms10071466
Bylund F, Collet E, Enfors S, Larsson G (1998) Substrate gradient formation in the large-scale bioreactor lowers cell yield and increases by-product formation. Bioprocess Eng 18:171–180. 10.1007/s004490050427 DOI: 10.1007/s004490050427
Bylund F, Guillard F, Enfors S, Trägârdh C, Larsson G (1999) Scale down of recombinant protein production: a comparative study of scaling performance. Bioprocess Eng 20:377–389. 10.1007/s004490050606 DOI: 10.1007/s004490050606
De S, Rebnegger C, Moser J, Tatto N, Graf A, Mattanovich D, Gasser B (2020) Pseudohyphal differentiation in Komagataella phaffii: investigating the FLO gene family. FEMS Yeast Res 20:1–15. 10.1093/femsyr/foaa044 DOI: 10.1093/femsyr/foaa044
Delvigne F, Lejeune A, Destain J, Thonart P (2006) Stochastic models to study the impact of mixing on a fed-batch culture of Saccharomyces cerevisiae. Biotechnol Prog 22:259–269. 10.1021/bp050255m DOI: 10.1021/bp050255m
Delvigne F, Zacchetti B, Fickers P, Fifani B, Roulling F, Lefebvre C, Neubauer P, Junne S (2018) Improving control in microbial cell factories: from single-cell to large-scale bioproduction. FEMS Microbiol Lett 365:1–11. 10.1093/femsle/fny236 DOI: 10.1093/femsle/fny236
Dubois M, Gilles K, Hamilton J, Rebers P, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356. 10.1021/ac60111a017 DOI: 10.1021/ac60111a017
Falcioni T, Manti A, Boi P, Canonico B, Balsamo M, Papa S (2006) Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry. Cytometry B Clin Cytom 70B:149–153. 10.1002/cyto.b.20097 DOI: 10.1002/cyto.b.20097
Francois J, Parrou J (2001) Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Lett 25(125):145. 10.1111/j.1574-6976.2001.tb00574.x DOI: 10.1111/j.1574-6976.2001.tb00574.x
Garcia-Ochoa F, Gomez E (2009) Bioreactor scale-up and oxygen transfer rate in microbial processes: an overview. Biotechnol Adv 27:153–176. 10.1016/j.biotechadv.2008.10.006 DOI: 10.1016/j.biotechadv.2008.10.006
Gunduz B, Berrios J, Binay B, Fickers P (2021) Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling. FEMS Yeast Res 21:1–14. 10.1093/femsyr/foab057 DOI: 10.1093/femsyr/foab057
Huang J, Wang Q, Bu W, Chen L, Yang Z, Zheng W, Li Y, Li J (2019) Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes. Bioengineered 10:150–161. 10.1080/21655979.2019.1614422 DOI: 10.1080/21655979.2019.1614422
Jordà J, de Jesus S, Peltier S, Ferrer P, Albiol J (2013) Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids. N Biotechnol 31:120–132. 10.1016/j.enzmictec.2014.10.006 DOI: 10.1016/j.enzmictec.2014.10.006
Jungo C, Marison I, von Stockar U (2007) Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures. J Biotechnol 130:236–246. 10.1016/j.jbiotec.2007.04.004 DOI: 10.1016/j.jbiotec.2007.04.004
Lara A, Galindo E, Ramírez O, Palomares L (2006) Living with heterogeneities in bioreactors. Mol Biotechnol 34:355–381. 10.1385/mb:34:3:355 DOI: 10.1385/mb:34:3:355
Lejeune A, Delvigne F, Thonart P (2013) Physiological response of yeast to process perturbations: a mini-bioreactor approach. Cerevisia 38:15–19. 10.1016/j.cervis.2013.04.004 DOI: 10.1016/j.cervis.2013.04.004
Levenspiel O (2012) The E and Eo Curves from pulse and step tracer experiments. Tracer Technology. Fluid Mechanics and Its Applications. Springer, New York, pp 11–24 DOI: 10.1007/978-1-4419-8074-8_3
Lorantfy B, Jazini M, Herwig C (2013) Investigation of the physiological response to oxygen limited process conditions of Pichia pastoris Mut+ strain using a two-compartment scale-down system. J Biosci Bioeng 116:371–379. 10.1016/j.jbiosc.2013.03.021 DOI: 10.1016/j.jbiosc.2013.03.021
Neubauer P, Junne S (2010) Scale-down simulators for metabolic analysis of large-scale bioprocesses. Curr Opin Biotechnol 21:114–121. 10.1016/j.copbio.2010.02.001 DOI: 10.1016/j.copbio.2010.02.001
Nienow A, Scott W, Hewitt C, Thomas C, Lewis G, Amanullah A, Kiss R, Meier S (2013) Scale-down studies for assessing the impact of different stress parameters on growth and product quality during animal cell culture. Cheml Eng Res Des 91:2265–2274. 10.1016/j.cherd.2013.04.002 DOI: 10.1016/j.cherd.2013.04.002
Noorman H, Morud K, Hjertager BH, Traegaardh C, Larsson G, Enfors SO (1993) CFD modeling and verification of flow and conversion in a 1 m3 bioreactor. BHR Group Conf Ser Publ 5:241–258
Oosterhuis N, Kossen N (1983) Oxygen transfer in a production scale bioreactor. Chem Eng Res Des 61:308–312
Oosterhuis N, Kossen N, Olivier A, Schenk E (1985) Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans. Biotechnol Bioeng 27:711–720. 10.1002/bit.260270521 DOI: 10.1002/bit.260270521
Osman J, Birch J, Varley J (2002) The response of GS-NS0 myeloma cells to single and multiple pH perturbations. Biotechnol Bioeng 79:398–407. 10.1002/bit.10198 DOI: 10.1002/bit.10198
Pfaffl M (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007. 10.1111/j.1365-2966.2012.21196.x DOI: 10.1111/j.1365-2966.2012.21196.x
Priyadi K, Lu C, Sutanto H (2019) Optimization of impeller design for stirred tank using computational fluid dynamics. IOP Conf Ser Mater Sci Eng 567:1–6. 10.1088/1757-899X/567/1/012032 DOI: 10.1088/1757-899X/567/1/012032
Rebnegger C, Graf A, Valli M, Steiger M, Gasser B, Maurer M, Mattanovich D (2014) In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response. Biotechnol J 9:511–525. 10.1002/biot.201300334 DOI: 10.1002/biot.201300334
Rebnegger C, Vos T, Graf A, Valli M, Pronk J, Daran-Lapujade P, Mattanovich D (2016) Pichia pastoris exhibits high viability and low maintenance-energy requirement at near-zero specific growth rates. Appl Environ Microbiol 82:AEM.00638-16. https://doi.org/10.1128/AEM.00638-16
Ronen M, Botstein D (2006) Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source. PNAS 103:389–394. 10.1073/pnas.0509978103 DOI: 10.1073/pnas.0509978103
Singh A, Narang A (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. Appl Microbiol Biotechnol 104:7801–7814 DOI: 10.1007/s00253-020-10793-8
Theron C, Berrios J, Steels S, Telek S, Lecler R, Rodriguez C, Fickers P (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. 10.1002/yea.3388 DOI: 10.1002/yea.3388
Theron C, Vandermies M, Telek S, Steels S, Fickers P (2020) Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B. Sci Rep 10:1–9. 10.1038/s41598-020-58683-3 DOI: 10.1038/s41598-020-58683-3
Vallejo J, Villa T (2013) Cell aggregations in yeasts and their applications. Appl Microbiol Biotechnol 97:2305–2318. 10.1007/s00253-013-4735-y DOI: 10.1007/s00253-013-4735-y
Velastegui E, Theron C, Berrios J, Fickers P (2019) Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris. Yeast 36:297–304. 10.1002/yea.3381 DOI: 10.1002/yea.3381
Vrábel P, van der Lans R, Cui Y, Luyben K (1999) Compartment model approach: mixing in large scale aerated reactors with multiple impellers. Chem Eng Res Des 77:291–302. 10.1205/026387699526223 DOI: 10.1205/026387699526223
Werringloer J (1978) Assay of formaldehyde generated during microsomal oxidation reactions. Methods Enzymol 297–302. https://doi.org/10.1016/s0076-6879(78)52031-4
Yan C, Xu X, Zhang X, Zhang Y, Zhang Y, Zhang Z (2018) Decreased rhamnose metabolic flux improved production of target proteins and cell flocculation in Pichia pastoris. Front Microbiol 9:1–11. 10.3389/fmicb.2018.01771 DOI: 10.3389/fmicb.2018.01771
Zepeda A, Figueroa C, Abdalla D, Maranhão A, Ulloa P, Pessoa A, Farías J (2014) Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris. Brazilian J Microbiol 45:475–483. 10.1590/S1517-83822014000200014 DOI: 10.1590/S1517-83822014000200014