Pichia pastoris (Komagataella sp.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli (EcPGA), lipase B from Candida antarctica (CaLB) and xylanase A from Thermomyces lanuginosus (TlXynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing EcPGA, which accumulated in the cells (50-70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing CaLB (20%) and TlXynA (27%). The proportion of cells with up-regulated UPR in CaLB-producing (35%) and TlXynA-producing (30%) strains was lower in comparison to the EcPGA-producing strain, and a smaller proportion of CaLB and TlXynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity.

Department of Biotechnology University of Chemistry and Technology Prague Prague Czechia

Department of Genetics and Microbiology Charles University Prague Czechia

Institute of Chemistry and Biotechnology School of Life Sciences and Facility Management Zurich University of Applied Sciences ZHAW Wädenswil Switzerland

Institute of Molecular Biotechnology Graz University of Technology Graz Austria

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czechia

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Ahmad M., Hirz M., Pichler H., Schwab H. (2014). Protein expression in PubMed DOI PMC

Alber A. B., Paquet E. R., Biserni M., Naef F., Suter D. M. (2018). Single live cell monitoring of protein turnover reveals intercellular variability and cell-cycle dependence of degradation rates. Mol. Cell. 71, 1079–1091. 10.1016/j.molcel.2018.07.023 PubMed DOI

Aw R., Barton G. R., Leak D. J. (2017). Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in PubMed DOI PMC

Broger T., Odermatt R. P., Huber P., Sonnleitner B. (2011). Real-time on-line flow cytometry for bioprocess monitoring. J. Biotechnol. 154, 240–247. 10.1016/j.jbiotec.2011.05.003 PubMed DOI

Cereghino J. L., Cregg J. M. (2000). Heterologous protein expression in the methylotrophic yeast PubMed DOI

Cox J. S., Shamu C. E., Walter P. (1993). Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73, 1197–1206. 10.1016/0092-8674(93)90648-A PubMed DOI

Cregg J. M., Cereghino J. L., Shi J., Higgins D. R. (2000). Recombinant protein expression in PubMed DOI

Damaso M. C., Almeida M. S., Kurtenbach E., Martins O. B., Pereira N, Jr, Andrade C. M., et al. (2003). Optimized expression of a thermostable xylanase from PubMed DOI PMC

Dragosits M., Stadlmann J., Graf A., Gasser B., Maurer M., Sauer M., et al. (2010). The response to unfolded protein is involved in osmotolerance of PubMed DOI PMC

Dudek J., Benedix J., Cappel S., Greiner M., Jalal C., Müller L., et al. (2009). Functions and pathologies of BiP and its interaction partners. Cell. Mol. Life Sci. 66, 1556–1569. 10.1007/s00018-009-8745-y PubMed DOI PMC

Edwards-Jones B., Aw R., Barton G. R., Tredwell G. D., Bundy J. G., Leak D. J. (2015). Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of PubMed DOI PMC

Gao B., Wang X., Shen Y. (2006). Studies on characters of immobilizing penicillin G acylase on a novel composite support PEI/SiO DOI

Gasser B., Maurer M., Rautio J., Sauer M., Bhattacharyya A., Saloheimo M., et al. (2007). Monitoring of transcriptional regulation in PubMed DOI PMC

Graf A., Gasser B., Dragosits M., Sauer M., Leparc G. G., Tüchler T., et al. (2008). Novel insights into the unfolded protein response using PubMed DOI PMC

Gruber K., Klintschar G., Hayn M., Schlacher A., Steiner W., Kratky C. (1998). Thermophilic xylanase from thermomyces lanuginosus: high-resolution X- ray structure and modeling studies. Biochemistry 37, 13475–13485. 10.1021/bi980864l PubMed DOI

Guerfal M., Ryckaert S., Jacobs P. P., Ameloot P., Van Craenenbroeck K., Derycke R., et al. (2010). The HAC1 gene from PubMed DOI PMC

Hesketh A. R., Castrillo J. I., Sawyer T., Archer D. B., Oliver S. G. (2013). Investigating the physiological response of PubMed DOI PMC

Hohenblum H., Borth N., Mattanovich D. (2003). Assessing viability and cell-associated product of recombinant protein producing PubMed DOI

Hohenblum H., Gasser B., Maurer M., Borth N., Mattanovich D. (2004). Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant PubMed DOI

Hyka P., Züllig T., Ruth C., Looser V., Meier C., Klein J., et al. (2010). Combined use of fluorescent dyes and flow cytometry to quantify the physiological state of PubMed DOI PMC

Jahic M., Wallberg F., Bollok M., Garcia P., Enfors S. O. (2003). Temperature limited fed-batch technique for control of proteolysis in PubMed DOI PMC

Juturu V., Wu J. C. (2018). Heterologous protein expression in PubMed DOI

Kacmar J., Gilbert A., Cockrell J., Srienc F. (2006). The cytostat: a new way to study cell physiology in a precisely defined environment. J. Biotechnol. 126, 163–72. 10.1016/j.jbiotec.2006.04.015 PubMed DOI

Khatri N. K., Gocke D., Trentmann O., Neubauer P., Hoffmann F. (2011). Single-chain antibody fragment production in PubMed DOI

Khmelinskii A., Keller P. J., Bartosik A., Meurer M., Barry J. D., Mardin B. R., et al. (2012). Tandem fluorescent protein timers for PubMed DOI

Krainer F. W., Dietzsch C., Hajek T., Herwig C., Spadiut O., Glieder A. (2012). Recombinant protein expression in PubMed DOI PMC

Lajoie P., Moir R. D., Willis I. M., Snapp E. L. (2012). Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells. Mol. Biol. Cell 23, 955–964. 10.1091/mbc.e11-12-0995 PubMed DOI PMC

Lin X. Q., Liang S. L., Han S. Y., Zheng S. P., Ye Y. R., Lin Y. (2013). Quantitative iTRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in PubMed DOI

Lin-Cereghino J., Wong W. W., Xiong S., Giang W., Luong L. T., Vu J., et al. (2005). Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast PubMed DOI PMC

Love K. R., Panagiotou V., Jiang B., Stadheim T. A., Love J. C. (2010). Integrated single-cell analysis shows PubMed DOI

Love K. R., Politano T. J., Panagiotou V., Jiang B., Stadheim T. A., Love J. C. (2012). Systematic single-cell analysis of PubMed DOI PMC

Madjid Ansari A., Majidzadeh -A K, Darvishi B., Sanati H., Farahmand L., Norouzian D., et al. (2017). Extremely low frequency magnetic field enhances glucose oxidase expression in PubMed DOI

Madzak C., Beckerich J.-M. (2006). A sensor of the unfolded protein response to study the stress induced in DOI

Marešová H., Palyzová A., Plačková M., Grulich M., Rajasekar V. W., Štěpánek V., et al. (2017). Potential of PubMed DOI

Mattanovich D., Gasser B., Hohenblum H., Sauer M. (2004). Stress in recombinant protein producing yeasts. J. Biotechnol. 113, 121–135. 10.1016/j.jbiotec.2004.04.035 PubMed DOI

Meehl M. A., Stadheim T. A. (2014). Biopharmaceutical discovery and production in yeast. Curr. Opin. Biotechnol. 30, 120–127. 10.1016/j.copbio.2014.06.007 PubMed DOI

Mellitzer A., Glieder A., Weis R., Reisinger C., Flicker K. (2012a). Sensitive high-throughput screening for the detection of reducing sugars. Biotechnol. J. 7, 155–162. 10.1002/biot.201100001 PubMed DOI

Mellitzer A., Weis R., Glieder A., Flicker K. (2012b). Expression of lignocellulolytic enzymes in PubMed DOI PMC

Mori K., Kawahara T., Yoshida H., Yanagi H., Yura T. (1996). Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells 1, 803–817. 10.1046/j.1365-2443.1996.d01-274.x PubMed DOI

Mori K., Ogawa N., Kawahara T., Yanagi H., Yura T. (1998). Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in PubMed DOI

Pédelacq J. D., Cabantous S., Tran T., Terwilliger T. C., Waldo G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88. 10.1038/nbt1172 PubMed DOI

Pfeffer M., Maurer M., Köllensperger G., Hann S., Graf A. B., Mattanovich D. (2011). Modeling and measuring intracellular fluxes of secreted recombinant protein in PubMed DOI PMC

Potgieter T. I., Kersey S. D., Mallem M. R., Nylen A. C., d'Anjou M. (2010). Antibody expression kinetics in glycoengineered PubMed DOI

Puxbaum V., Mattanovich D., Gasser B. (2015). Quo vadis? The challenges of recombinant protein folding and secretion in PubMed DOI

Rebnegger C., Graf A. B., Valli M., Steiger M. G., Gasser B., Maurer M., et al. (2014). in PubMed DOI PMC

Rebnegger C., Vos T., Graf A. B., Valli M., Pronk J. T., Daran-Lapujade P., et al. (2016). PubMed DOI PMC

Reséndiz-Cardiel G., Arroyo R., Ortega-López J. (2017). Expression of the enzymatically active legumain-like cysteine proteinase TvLEGU-1 of PubMed DOI

Resina D., Bollók M., Khatri N. K., Valero F., Neubauer P., Ferrer P. (2007). Transcriptional response of P-pastoris in fed-batch cultivations to PubMed DOI PMC

Roth G., Vanz A. L., Lünsdorf H., Nimtz M., Rinas U. (2018). Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing PubMed DOI PMC

Samuel P., Prasanna Vadhana A. K., Kamatchi R., Antony A., Meenakshisundaram S. (2013). Effect of molecular chaperones on the expression of PubMed DOI

Sjöblom M., Lindberg L., Holgersson J., Rova U. (2012). Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density DOI

Sobotková L., Štěpánek V., Plháčková K., Kyslík P. (1996). Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3(pKA18). Enzyme Microb. Technol. 19, 389–397. 10.1016/S0141-0229(96)00052-X DOI

Summpunn P., Jomrit J., Panbangred W. (2018). Improvement of extracellular bacterial protein production in PubMed DOI

Theron C. W., Berrios J., Delvigne F., Fickers P. (2018). Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella PubMed DOI

Tredwell G. D., Aw R., Edwards-Jones B., Leak D. J., Bundy J. G. (2017). Rapid screening of cellular stress responses in recombinant PubMed DOI PMC

Uppenberg J., Hansen M. T., Patkar S., Jones T. A. (1994). The sequence, crystal structure determination and refinement of two crystal forms of lipase B from PubMed DOI

Vanz A. L., Nimtz M., Rinas U. (2014). Decrease of UPR- and ERAD-related proteins in PubMed DOI PMC

Vogl T., Thallinger G. G., Zellnig G., Drew D., Cregg J. M., Glieder A., et al. (2014). Towards improved membrane protein production in PubMed DOI

Wang J., Li Y., Liu D. (2016). Improved production of PubMed DOI PMC

Wang X. D., Jiang T., Yu X. W., Xu Y. (2017). Effects of UPR and ERAD pathway on the prolyl endopeptidase production in PubMed DOI

Weis R., Luiten R., Skranc W., Schwab H., Wubbolts M., Glieder A. (2004). Reliable high-throughput screening with PubMed DOI

Yu X. W., Sun W. H., Wang Y. Z., Xu Y. (2017). Identification of novel factors enhancing recombinant protein production in multi-copy Komagataella phaffii based on transcriptomic analysis of overexpression effects. Sci. Rep. 7:16249. 10.1038/s41598-017-16577-x PubMed DOI PMC

Zahrl R. J., Mattanovich D., Gasser B. (2018). The impact of ERAD on recombinant protein secretion in pichia pastoris (Syn komagataella spp.). Microbiol. 164, 453–463. 10.1099/mic.0.000630 PubMed DOI

Zahrl R. J., Peña D. A., Mattanovich D., Gasser B. (2017). Systems biotechnology for protein production in PubMed DOI

Zhong Y., Yang L., Guo Y., Fang F., Wang D., Li R., et al. (2014). High-temperature cultivation of recombinant PubMed DOI PMC

Zhu T., Guo M., Sun C., Qian J., Zhuang Y., Chu J., et al. (2009). A systematical investigation on the genetic stability of multi-copy PubMed DOI

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins