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 Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 98, 5301–5317. 10.1007/s00253-014-5732-5 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 Pichia pastoris. Appl. Microbiol. Biotechnol. 101, 5045–5058. 10.1007/s00253-017-8317-2 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 Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66. 10.1111/j.1574-6976.2000.tb00532.x 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 Pichia pastoris. Mol. Biotechnol. 16, 23–52. 10.1385/MB:16:1:23 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 Thermomyces lanuginosus in Pichia pastoris. Appl. Env. Microbiol. 69, 6064–6072. 10.1128/AEM.69.10.6064-6072.2003 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 Pichia pastoris. BMC Genomics 11:207. 10.1186/1471-2164-11-207 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 Pichia pastoris. PLoS ONE 10:e0119637. 10.1371/journal.pone.0119637 PubMed DOI PMC

Gao B., Wang X., Shen Y. (2006). Studies on characters of immobilizing penicillin G acylase on a novel composite support PEI/SiO2. Biochem. Eng. J. 28, 140–147. 10.1016/j.bej.2005.10.007 DOI

Gasser B., Maurer M., Rautio J., Sauer M., Bhattacharyya A., Saloheimo M., et al. . (2007). Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions. BMC Genomics 8:179. 10.1186/1471-2164-8-179 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 Pichia pastoris specific DNA microarrays. BMC Genomics 9:390. 10.1186/1471-2164-9-390 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 Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb. Cell Fact. 9:49. 10.1186/1475-2859-9-49 PubMed DOI PMC

Hesketh A. R., Castrillo J. I., Sawyer T., Archer D. B., Oliver S. G. (2013). Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures. Appl. Microbiol. Biotechnol. 97, 9747–9762. 10.1007/s00253-013-5186-1 PubMed DOI PMC

Hohenblum H., Borth N., Mattanovich D. (2003). Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J. Biotechnol. 102, 281–290. 10.1016/S0168-1656(03)00049-X 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 Pichia pastoris. Biotechnol. Bioeng. 85, 367–375. 10.1002/bit.10904 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 Pichia pastoris during the production of heterologous proteins in high-cell-density fed-batch cultures. Appl. Env. Microbiol. 76, 4486–4496. 10.1128/AEM.02475-09 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 Pichia pastoris bioreactor cultures. Microb. Cell Fact. 2:6. 10.1186/1475-2859-2-6 PubMed DOI PMC

Juturu V., Wu J. C. (2018). Heterologous protein expression in Pichia pastoris: latest research progress and applications. ChemBioChem 19, 7–21. 10.1002/cbic.201700460 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 Pichia pastoris: benefits of prolonged pre-induction glycerol feeding. Biotechnol. J. 6, 452–462. 10.1002/biot.201000193 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 in vivo analysis of protein dynamics. Nat. Biotechnol. 30, 708–714. 10.1038/nbt.2281 PubMed DOI

Krainer F. W., Dietzsch C., Hajek T., Herwig C., Spadiut O., Glieder A. (2012). Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb. Cell Fact. 11:22. 10.1186/1475-2859-11-22 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 Pichia pastoris. J. Proteomics 91, 58–72. 10.1016/j.jprot.2013.06.031 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 Pichia pastoris. Biotechniques 38, 44–48. 10.2144/05381BM04 PubMed DOI PMC

Love K. R., Panagiotou V., Jiang B., Stadheim T. A., Love J. C. (2010). Integrated single-cell analysis shows Pichia pastoris secretes protein stochastically. Biotechnol. Bioeng. 106, 319–325. 10.1002/bit.22688 PubMed DOI

Love K. R., Politano T. J., Panagiotou V., Jiang B., Stadheim T. A., Love J. C. (2012). Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity. PLoS ONE 7:e37915. 10.1371/journal.pone.0037915 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 Pichia pastoris GS115. Enzyme Microb. Technol. 98, 67–75. 10.1016/j.enzmictec.2016.12.011 PubMed DOI

Madzak C., Beckerich J.-M. (2006). A sensor of the unfolded protein response to study the stress induced in Yarrowia lipolytica strains by the production of heterologous proteins. Microb. Cell Fact. 5:P5 10.1186/1475-2859-5-S1-P5 DOI

Marešová H., Palyzová A., Plačková M., Grulich M., Rajasekar V. W., Štěpánek V., et al. . (2017). Potential of Pichia pastoris for the production of industrial penicillin G acylase. Folia Microbiol. (Praha). 62, 417–424. 10.1007/s12223-017-0512-0 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 Pichia pastoris. Microb. Cell Fact. 11:61. 10.1186/1475-2859-11-61 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 Saccharomyces cerevisiae. J. Biol. Chem. 273, 9912–9920. 10.1074/jbc.273.16.9912 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 Pichia pastoris with a novel 34S labeling procedure. Microb. Cell Fact. 10:47. 10.1186/1475-2859-10-47 PubMed DOI PMC

Potgieter T. I., Kersey S. D., Mallem M. R., Nylen A. C., d'Anjou M. (2010). Antibody expression kinetics in glycoengineered Pichia pastoris. Biotechnol. Bioeng. 106, 918–927. 10.1002/bit.22756 PubMed DOI

Puxbaum V., Mattanovich D., Gasser B. (2015). Quo vadis? The challenges of recombinant protein folding and secretion in Pichia pastoris. Appl. Microbiol. Biotechnol. 99, 2925–2938. 10.1007/s00253-015-6470-z PubMed DOI

Rebnegger C., Graf A. B., Valli M., Steiger M. G., Gasser B., Maurer M., et al. . (2014). in Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response. Biotechnol. J. 9, 511–525. 10.1002/biot.201300334 PubMed DOI PMC

Rebnegger C., Vos T., Graf A. B., Valli M., Pronk J. T., Daran-Lapujade P., et al. . (2016). Pichia pastoris exhibits high viability and low maintenance-energy requirement at near-zero specific growth rates. Appl. Environ. Microbiol. 82, 4570–4583. 10.1128/AEM.00638-16 PubMed DOI PMC

Reséndiz-Cardiel G., Arroyo R., Ortega-López J. (2017). Expression of the enzymatically active legumain-like cysteine proteinase TvLEGU-1 of Trichomonas vaginalis in Pichia pastoris. Protein Expr. Purif. 134, 104–113. 10.1016/j.pep.2017.04.007 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 Rhizopus oryzae lipase production reveals UPR induction. Microb. Cell Fact. 6:21. 10.1186/1475-2859-6-21 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 Pichia pastoris. Microb. Cell Fact. 17:123. 10.1186/s12934-018-0970-3 PubMed DOI PMC

Samuel P., Prasanna Vadhana A. K., Kamatchi R., Antony A., Meenakshisundaram S. (2013). Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris. Microbiol. Res. 168, 615–620. 10.1016/j.micres.2013.06.007 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 Pichia pastoris bioreactor cultivations. Adv. Biosci. Biotechnol. 3, 238–248. 10.4236/abb.2012.33033 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 Pichia pastoris by co-expression of endoplasmic reticulum residing GroEL–GroES. J. Biosci. Bioeng. 125, 268–274. 10.1016/j.jbiosc.2017.09.007 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 (Pichia) pastoris. Appl. Microbiol. Biotechnol. 102, 63–80. 10.1007/s00253-017-8612-y 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 Pichia pastoris strains using metabolite profiling. J. Ind. Microbiol. Biotechnol. 44, 413–417. 10.1007/s10295-017-1904-5 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 Candida antarctica. Structure 2, 293–308. 10.1016/S0969-2126(00)00031-9 PubMed DOI

Vanz A. L., Nimtz M., Rinas U. (2014). Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures. Microb. Cell Fact. 13:10. 10.1186/1475-2859-13-23 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 Pichia pastoris: general and specific transcriptional response to membrane protein overexpression. N. Biotechnol. 31, 538–552. 10.1016/j.nbt.2014.02.009 PubMed DOI

Wang J., Li Y., Liu D. (2016). Improved production of Aspergillus usamii endo-β-1,4-Xylanase in Pichia pastoris via combined strategies. Biomed Res. Int. 2016:3265895. 10.1155/2016/3265895 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 Pichia pastoris by controlling of nitrogen source. J. Ind. Microbiol. Biotechnol. 44, 1053–1063. 10.1007/s10295-017-1938-8 PubMed DOI

Weis R., Luiten R., Skranc W., Schwab H., Wubbolts M., Glieder A. (2004). Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res. 5, 179–189. 10.1016/j.femsyr.2004.06.016 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 Pichia pastoris. FEMS Yeast Res. 17, 1–15. 10.1093/femsyr/fox068 PubMed DOI

Zhong Y., Yang L., Guo Y., Fang F., Wang D., Li R., et al. . (2014). High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10. Microb. Cell Fact. 13:163. 10.1186/s12934-014-0163-7 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 Pichia pastoris strains. Biotechnol. Lett. 31, 679–684. 10.1007/s10529-009-9917-4 PubMed DOI

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