Neng Herawati, Arizah Kusumawati, Adi Santoso
| Abstract views: 1073 | PDF views: 1276


Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viruses so it is safer and simplifies the downstream processes in bioproduction. The level of endogenous protein in the low supernatant allows Pichia to cultivate with a high volumetric productivity, therefore the process of protein production becomes very economical. This review provides an overview of several things that must be considered in utilizing P. pastoris as an expression system including the selection of vectors, strains, vector integration mechanisms into the genome, glycosylation processes, and applications in industry.


Pichia pastoris, expression system, glycosylation, vector, AOX promoter

Full Text:



Ahmad, M., Hirz, M., Pichler, H. and Schwab, H., 2014. Protein expression in Pichia pastoris: Recent achievement and perspectives for heterologous protein production. Applied Microbiology and Biotechnology, 98, pp. 5301–5317.

Anh, M.T., Thanh, N.T., Cong, N., Xuan, M.H.T., Cao, T.N., Minh, T.T., Linh, T.T., Stephanie, P.C., Roslyn, M.B. and Hieu, T.V., 2017. Pichia pastoris versus Saccharomyces cerevisiae: A case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor. BMC Res Notes, 10(148), pp 1–8.

Aw, R. and Polizzi, K.M., 2013. Can too many copies spoil the broth. Microbial Cell factories, 12(128), pp. 1–9.

Ballou, L., Hernandez, L.M., Alvarado, E. and Ballaou, C.E., 1990. Revision of the oligosaccharide structures of yeast carboxy peptidase Y. Proceedings of the National Academy of Sciences of the United States of America, 87, pp. 3368–3372.

Brierley, R.A., 1998. Secretion of recombinant human insulin like growth factor I (IGF-1). Methods in Molecular Biology, 103, pp. 149–177.

Cereghino, J.l. and Cregg, J.M., 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24, pp. 45–66.

Ciofalo, V., Barton, N., Kreps, J., Coats, I. and Shanahan, D., 2006. Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil. Regulatory Toxicology and Pharmacology, 45(1), pp. 1–8.

Cregg, J. and Tolstorukov, I., 2012. P. pastoris ADH promoter and use thereof to direct expression of proteins. United States Patent US, No. 8222386.

Cregg, J.M., Barringer, K.J., Hessler, A.Y. and Madden K.R., 1985. Pichia pastoris as a host system for transformations. Molecular and Cellular Biology, 5(12), pp. 3376–3385.

Damasceno, L.M., Huang, C.J. and Batt, C.A., 2012. Protein seceretion in Pichia pastoris and advances in protein production. Applied Microbiology and Biotechnology, 93, pp. 31–39.

Davis, B.G., 2002. Synthesis of glycoproteins. Chemical Reviews, 102(2), pp. 579–601.

De Almeida, J.R., de Moraes, L.M. and Torres, F.A., 2005. Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast, 22, pp. 725–737.

De Pourcq, K., De Schutter, K. and Callewaert, N., 2010. Engineering of glycosylation in yeast and other fungi: Current state and perspectives. Applied Microbiology and Biotechnology, 87, pp. 1617–1631.

De Schutter, K., Lin, Y.C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouzé, P., Van de Peer, Y. and Callewaert, N., 2009. Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology, 27(6), pp. 561–566.

Du, M., Battles, M.B. and Nett, J.H., 2012. A color-based stable multi-copy integrant selection system for Pichia pastoris using the attenuated ADE1 and ADE2 genes as auxotrophic markers. Bioengineered, 3, pp.32–37.

Gasser, B., Prielhofer, R., Marx, H., Maurer, M., Nocon, J., Steiger, M., Puxbaum, V., Sauer, M. and Mattanovich, D., 2013. Pichia pastoris: Protein production host and model organism for biomedical research. Future Microbiology, 8, pp. 191–208.

Hamilton, S.R. and Gerngross, T.U., 2007. Glycosylation engineering in yeast: The advent of fully humanized yeast. Current Opinion in Biotechnology, 18(5), pp. 387–392.

Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H. and Choi, B.K., 2006. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science, 313, pp. 1441–1443.

Hartner, F.S., Ruth, C., Langenegger, D., Johnson, S.N., Hyka, P., Lin-Cereghino, G.P., Lin-Cereghino, J., Kovar, K., Cregg, J.M. and Glieder, A., 2008. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Research, 36(12), e76, pp. 1–15.

Helenius, A. and Aebi, M., 2001. Intracellular function of N-linked glycans. Science, 291(5512), pp. 2364–2369.

Hossler, P., Mulukutla, B.C. and Hu, W.S., 2007. Systems analysis of N-glycan processing in mammalian cells. PLoS ONE, 2(1), e713, pp. 1–17.

Inan, M. and Meagher, M.M., 2001. Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. Journal of Bioscience and Bioengineering, 92(6), pp. 585–589.

Ingund, R.R., 2012. Improvement of Pichia pastoris AOX1 Promoter Expression System. Institute fur Molekulare Biotechnologie. Dissertation, Technische Universitat, Graz.

Invitrogen, 2001. EasyselectTM Pichia expression kit: A manual of methods for expression of recombinant proteins using pPICZ and pPICZ? in Pichia pastoris. Catalog no. K1740-01.

Jahic, M., Rotticci, M.J., Martinelle, M., Hult, K. and Enfors, S.O., 2002. Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein. Bioprocess and Biosystems Engineering, 24, pp. 385–393.

Julien, C. 2006. Production of humanlike recombinants proteins in Pichia pastoris: From expression vector to fermentation strategy. Bioprocess Technical, 4(1)pp. 22–31.

Jun, Y., Cai, H., Liu, J., Zeng, M., Chen, J., Cheng, Q. and Zhang, L. 2018. Controlling AOX1 promoter strength in Pichia pastoris by manipulating poly (dA:dT) tracts. Scientific Reports, 8(1401), pp. 1–11. doi:10.1038/s41598-018-19831-y.

Jungoh, A., Jiyeon, H., Hyeokweon, L., Myongsoo, P., Eungyo, L., Chunsuk, K., Euisung, C., Joonki, J. and Hongweon, L., 2007. Translation elongation factor 1-? gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Applied Microbiology and Biotechnology, 74, pp. 601–608.

Krasovska, O.S., Stasyk, O.G., Nahorny, V.O., Stasyk, O.V., Granovski, N., Kordium, V.A., Vozianov, O.F. and Sibirny, A.A., 2007. Glucose induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression. Biotechnology and Bioengineering, 97, pp. 858–870.

Küberl, A., Schneider, J., Thallinger, G.G., Anderl, I., Wibberg, D., Hajek, T., Jaenicke, S., Brinkrolf, K., Goesmann, A., Szczepanowski, R., Pühler, A., Schwab, H., Glieder, A. and Pichler, H., 2011. High-quality genome sequence of Pichia pastoris CBS7435. Journal of Biotechnology, 154(4), pp. 312–320.

Kurtzman, C.P., 2009. Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. Journal of Industrial Microbiology & Biotechnology, 36(11), pp. 1435–1438.

Li, P., Anumanthan, A., Gao X.G., Ilangovan, K., Suzara, V.V., Düzgüne?, N. and Renugopalakrishnan, V., 2007. Expression of recombinant Proteins in Pichia pastoris. Biotechnology and Applied Biochemistry, 142, pp. 105–12

Li, P., Anumanthan, A., Gao, X.G., Ilangovan, K., Suzana, V.V., Duzgunes, N. and Renugopa, L.V., 2007. Expression of recombinant protein in Pichia pastoris. Biotechnology and Applied Biochemistry, 142, pp. 105–124.

Liang, S., Lin, Y., Chengjuan, Z. and Ye, Y., 2013. Identification and characterization of PGCW14: A novel, strong constitutive promoter of Pichia pastoris. Biotechnology Letters, 35(11), pp. 1865–1871.

Lin Cereghino, J. and Lin Cereghino, G.P., 2008. Vectors and strains for expression. Methods in Molecular Biology, 389, pp. 11–26.

Macauley, P.S., Fazenda, M.L., McNeil, B. and Harvey, L.M., 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast, 22, pp. 249–270.

Marx, H., Mecklenbrauker A., Gasser, B., Sauer, M. and Mattanovich, D., 2009. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Research, 9(8), pp. 1260–1270.

Matasci, M., Hacker, D.L., Baldi, L. and Wurm, F.M., 2008. Protein therapeutics recombinant therapeutic protein production in cultivated mammalian cells: Current status and future prospects. In: Drug Discovery Today: Technologies, 5, pp. 37–42.

Yu, X.W., Sha C., Guo, Y.L., Xiao, R. and Xu, Y., 2013. High level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production. Biotechnol Biofuels, 6(29), pp. 1–12. doi:10.1186/17546834-6-29.

Zhang, A.L., Luo, J.X., Zhang, T.Y., Pan, Y.W., Tan, Y.H., Fu, C.Y. and Tu, F.Z., 2009. Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep, 36, pp. 1611–1619.

Zhang, P., Zhang, W., Zhou, X., Bai, P., Cregg, J.M. and Zhang, Y., 2010. Catabolite repressionof Aox in Pichia pastoris is dependent on hexose transporter PpHxt1 and pexophagy. Appl Environ Microbiol, 76, pp. 6108–6118.


  • There are currently no refbacks.