Fed-batch cultivation and operational conditions for the production of a recombinant anti-amoebic vaccine in Pichia pastoris system.

  • S. L. Martínez-Hernández
  • M. A. Marin-Muñoz
  • J. Ventura-Juarez
  • J. Jauregui
Keywords: Methanol limited feed, recombinant vaccine, alcohol oxidase promoter, stirred tank bioreactor, shake flask


It was developed a fed-batch bioprocess to produce a recombinant vaccine against Entamoeba histolytica under operational conditions attainable to large scale bioprocesses. We have produced this recombinant protein in shake flask and stirred tank bioreactor. Initial results in shake flask cultures under different methanol concentration of 0.5, 1.5 and 3% (v/v) produced extracellular protein at quantities of 10, 22 and 33 μg/mL, respectively. Then a scale-up process was performed from shake flask to fermenter by keeping similar volumetric power supply (P/V). The operational conditions were set up in fermenter as those used at commercial scale and supply of pure oxygen was avoided to keep the scalability of the bioprocess. After the scale-up process, the production of the recombinant protein reached 0.43 mg/mL, an improvement in production of 12 times, although the methanol and oxygen limited conditions observed. Maximum volumetric productivity of 3.75 mg/L h was achieved in fermenter cultures against 263.75 µg/L h reached in shake flask. Besides the limited conditions in methanol and oxygen, the yields obtained from the bioprocess were comparable to those observed in Mut+ strains previously reported, then saturated methanol conditions are not necessary to compensate limited oxygen conditions.


Ahmad, M., Hirz, M., Pichler, H. and Schwab, H. (2014). Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology 98, 5301-5317. https://www.academia.edu/29845078/Protein_expression_in_Pichia_pastoris_recent_achievements_and_perspectives_for_heterologous_protein_production

Bansal, D., Malla, N. and Mahajan, R. C. (2006). Drug resistance in amoebiasis. Indian Journal of Medical Research 123, 115-118. http://medind.nic.in/iby/t06/i2/ibyt06i2p123.pdf

Bruckner, D. A. (1992). Amebiasis. Clinical Microbiology Reviews 5, 356-369. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC358254/

Büchs, J. (2001). Introduction to advantages and problems of shaken cultures. Biochemical Engineering Journal 7, 91-98. https://www.sciencedirect.com/science/article/pii/S1369703X00001066

Büchs, J., Lotter, S. and Milbradt, C. (2001). Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors. Biochemical Engineering Journal 7, 135-141. https://www.sciencedirect.com/science/article/pii/S1369703X00001133

Büchs, J., Maier, U., Milbradt, C. and Zoels, B. (2000a). Power consumption in shaking flasks on rotary shaking machines: I. Power consumption measurement in unbaffled flasks at low liquid viscosity. Biotechnology and Bioengineering 68, 589-593. https://onlinelibrary.wiley.com/doi/abs/10.1002/(SICI)1097-0290(20000620)68:6%3C589::AID-BIT1%3E3.0.CO%3B2-J

Büchs, J., Maier, U., Milbradt, C. and Zoels, B. (2000b). Power consumption in shaking flasks on regimes in unbaffled flasks at elevated liquid viscosity. Biotechnology and Bioengineering 68, 594-601. https://onlinelibrary.wiley.com/doi/abs/10.1002/(SICI)1097-0290(20000620)68:6%3C594::AID-BIT2%3E3.0.CO%3B2-U

Charoenrat, T., Ketudat-Cairns, M., Stendahl-Andersen, H., Jahic, M. and Enfors, S. O. (2005). Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess and Biosystems Engineering 27, 399-406. https://core.ac.uk/download/pdf/70930822.pdf

Chrast, L., Chaloupkova, R. and Damborsky, J. (2018). Gram-scale production of recombinant microbial enzymes in shake flasks. FEMS Microbiology Letters 365, fnx265. https://academic.oup.com/femsle/article/365/2/fnx265/4693837

Gamboa-Suasnavart, R. A., Marín-Palacio, L. D., Martínez-Sotelo, J. A., Espitia, C., Servín-González, L., et al. (2013). Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis. World Journal of Microbiology and Biotechnology 29, 1421-1429. https://link.springer.com/article/10.1007/s11274-013-1305-5

Gamboa-Suasnavart, R.A., Marín-Palacio, L. D., López-Griego, L., Córdova-Aguilar, M. S., Valdez-Cruz, N.A., et al. (2019). Volumetric power input as a reliable parameter for scale-up from shake flask to stirred-tank bioreactor: production of a recombinant glycoprotein by Streptomyces lividans. Revista Mexicana de Ingeniería Química 18, 1085-1099. http://www.rmiq.org/ojs311/index.php/rmiq/article/view/363

García-Ochoa, F. and Gomez, E. (2009). Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview. Biotechnology Advances 27, 153-176. https://www.sciencedirect.com/science/article/pii/S0734975008001079

Goletz, T. J., Klimpel, K. R., Leppla, S. H., Keith, J. M. and Berzofsky, J. A. (1997). Delivery of antigens to the MHC class I pathway using bacterial toxins. Human Immunology 54, 129-136. https://www.sciencedirect.com/science/article/abs/pii/S0198885997000815

Gómez-Sánchez, C. E., Martínez-Trujillo, A. and Aguilar-Osorio, G. (2012). Oxygen transfer coefficient and the kinetic parameters of exo-polygalacturonase production by Aspergillus flavipes FP-500 in shake flasks and bioreactor. Letters in Applied Microbiology 55, 444-452. https://onlinelibrary.wiley.com/doi/full/10.1111/j.1472-765x.2012.03313.x

Gupta, A. and Rao, G. (2003). A study of oxygen transfer in shake flasks using a non‐invasive oxygen sensor. Biotechnology and Bioengineering 84, 351-358. https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.10740

Gurramkonda, C., Adnan, A., Gäbel, T., Lünsdorf, H., Ross, A., et al. (2009). Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. Microbial Cell Factories 8, 13. https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-8-13

Jahic, M., Gustavsson, M., Jansen, A., Martinelle, M. and Enfors, S. (2003). Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. Journal of Biotechnology 102, 45-53. https://www.sciencedirect.com/science/article/pii/S0168165603000038

Khatri, N. K. and Hoffmann, F. (2006). Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnology and Bioengineering 93, 871–879. https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.20773

Klöckner, W. and Büchs, J. (2012). Advances in shaking technologies. Trends in Biotechnology 30, 307-314. https://www.cell.com/trends/biotechnology/fulltext/S0167-7799(12)00031-5

Kuhn, J., Müller, H., Salzig, D. and Czermak, P. (2015). A rapid method for an offline glycerol determination during microbial fermentation. Electronic Journal of Biotechnology 18, 252-255. https://www.sciencedirect.com/science/article/pii/S0717345815000226

Lara, R. A. (2011). Recombinant protein production in Escherichia coli. Revista Mexicana de Ingeniería Química 10, 209-223. http://www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S1665-27382011000200006

Lee, C. Y., Lee, S. J., Jung, K. H., Katoh, S. and Lee, E. K. (2003). High dissolved oxygen tension enhances heterologous protein expression by recombinant Pichia pastoris. Process Biochemistry 38, 1147-1154. https://www.sciencedirect.com/science/article/pii/S0032959202002807

Liao, C., W., Chen, C., A., Lee, C., N., Su, Y., N., Chang, M., C., et al. (2005). Fusion protein vaccine by domains of bacterial exotoxin linked with a tumor antigen generates potent immunologic responses and antitumor effects. Cancer Research 65, 9089-9098. http://cancerres.aacrjournals.org/content/65/19/9089

Looser, V., Brühlmann, B., Bumbak, F., Stenger, C., Costa, M., Camattari, A., et al. (2015). Cultivation strategies to enhance productivity of Pichia pastoris: A review. Biotechnology advances 33, 1177-1193. https://www.sciencedirect.com/science/article/pii/S0734975015300070

López-Pérez, M. and Viniegra-González, G. (2017). Differential toxicity caused by methanol on the growth of Pichia pastoris cultured in solid-state and in submerged fermentation. Revista Mexicana de Ingeniería Química 16, 735-743. http://www.redalyc.org/resumen.oa?id=62053304004

Maier, U., Losen, M. and Büchs, J. (2004). Advances in understanding and modeling the gas–liquid mass transfer in shake flasks. Biochemical Engineering Journal 17, 155-167. https://www.sciencedirect.com/science/article/pii/S1369703X03001748

Mann, B. J. and Lockhart, L. A. (1998). Molecular analysis of the Gal/GalNAc adhesin of Entamoeba histolytica. Journal of Eukaryotic Microbiology 45, 13S-16S. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1550-7408.1998.tb04518.x

Markošová, K., Weignerova, L., Rosenberg, M., Křen, V. and Rebroš, M. (2015). Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris MutS strain. Frontiers in Microbiology 6, 1140. https://www.readcube.com/articles/10.3389/fmicb.2015.01140

Martínez-Hernández, S. L., Cervantes-García, D., Muñoz-Ortega, M., Aldaba-Muruato, L. R., Loera-Muro, V. M., et al. (2017). An anti-amoebic vaccine: generation of the recombinant antigen LC3 from Entamoeba histolytica linked to mutated exotoxin A (PEΔIII) via the Pichia pastoris system. Biotechnology Letters 39, 1149-1157. https://www.ncbi.nlm.nih.gov/pubmed/28470625

Neubauer, P. and Junne, S. (2016). Scale‐up and scale‐down methodologies for bioreactors. In: Bioreactors: Design, Operation and Novel Applications, (C. F. Mandenius, ed.), Pp. 323-354. Wiley-VCH Verlag GmbH and Co. KGaA. https://onlinelibrary.wiley.com/doi/10.1002/9783527683369.ch11

Pérez-Martínez, A. S., Barba de la Rosa, A. P., De León-Rodríguez, A. (2014). Heterologous expression of Trichoderma atroviride endochitinase ech42 in Pichia pastoris at low and high dissolved oxygen tensions. Revista Mexicana de Ingeniería Química 13, 93-101. http://www.scielo.org.mx/scielo.php?script=sci_abstract&pid=S1665-27382014000100008&lng=es&nrm=iso

Peter, C. P., Suzuki, Y., Rachinskiy, K., Lotter, S. and Büchs, J. (2006). Volumetric power consumption in baffled shake flasks. Chemical Engineering Science 61, 3771-3779. https://www.sciencedirect.com/science/article/pii/S0009250905009632

Petri, W. A., Haque, R. and Mann, B. J. (2002). The bittersweet interface of parasite and host: Lectin-carbohydrate interactions during human invasion by the parasite Entamoeba histolytica. Annual Review of Microbiology 56, 39-64. https://www.annualreviews.org/doi/abs/10.1146/annurev.micro.56.012302.160959

Potvin, G., Ahmad, A. and Zhang, Z. (2012). Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review. Biochemical Engineering Journal 64, 91-105. https://www.sciencedirect.com/science/article/pii/S1369703X10002184

Reyes, C., Peña, C. and Galindo, E. (2003). Reproducing shake flasks performance in stirred fermentors: production of alginates by Azotobacter vinelandii. Journal of Biotechnology 105, 189-198.

Rocha-Valadez, A., J., Estrada, M., Galindo, E. and Serrano-Carreón, L. (2006). From shake flasks to stirred fermentors: Scale-up of an extractive fermentation process for 6-pentyl-α-pyrone production by Trichoderma harzianum using volumetric power input. Process Biochemistry 41, 1347-1352. https://www.sciencedirect.com/science/article/pii/S1359511306000195

Santana, C., Anjo, S. I. and Manadas, B. (2016). Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach. PROTEOMICS 16, 1847-1851. https://onlinelibrary.wiley.com/doi/abs/10.1002/pmic.201600024

Seletzky, J. M., Noak, U., Fricke, J., Welk, E., Eberhard, W., et al. (2007). Scale‐up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH. Biotechnology and Bioengineering 98, 800-811. https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.21359

Trentmann, O., Khatri, N. K. and Hoffmann, F. (2004). Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnology Progress 20, 1766–1775.

Trujillo-Roldán, M. A., Valdez-Cruz, N. A., Gonzalez-Monterrubio, C. F., Acevedo-Sánchez, E. V., Martínez-Salinas, C., et al. (2013). Scale-up from shake flasks to pilot-scale production of the plant growth-promoting bacterium Azospirillum brasilense for preparing a liquid inoculant formulation. Applied Microbiology and Biotechnology 97, 9665-9674. https://link.springer.com/article/10.1007/s00253-013-5199-9

Van Suijdam, J. C., Kossen, N. W. F. and Joha, A. C. (1978). Model for oxygen transfer in a shake flask. Biotechnology and Bioengineering 20, 1695-1709. https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.260201102

Vieira Gomes, A., Souza Carmo, T., Silva Carvalho, L., Mendonça Bahia, F. and Parachin, N. (2018). Comparison of yeasts as hosts for recombinant protein production. Microorganisms 6, :38. https://www.mdpi.com/2076-2607/6/2/38
How to Cite
Martínez-Hernández, S., Marin-Muñoz, M., Ventura-Juarez, J., & Jauregui, J. (2019). Fed-batch cultivation and operational conditions for the production of a recombinant anti-amoebic vaccine in Pichia pastoris system. Revista Mexicana De Ingeniería Química, 19(2), 691-705. https://doi.org/10.24275/rmiq/Bio725