Pichia pastoris

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Pichia pastoris
Scientific classification
Binomial name
Pichia pastoris
(Guillierm.) Phaff 1956

Pichia pastoris is a species of methylotrophic yeast. Pichia is widely used for protein production using recombinant DNA techniques. Hence it is used in biochemical and genetic research in academia and the biotechnical industry. Pichia pastoris is the common name used for the expression system, but the species was latter reassigned as Komagataella phaffii.[1]

P. pastoris as an expression system[edit]

P. pastoris is frequently used as an expression system for the production of proteins. Several properties make P. pastoris suited for this task: P. pastoris has a high growth rate and is able to grow on a simple, inexpensive medium. P. pastoris can grow in either shake flasks or a fermenter, which makes it suitable for both small- and large-scale production.

P. pastoris has two alcohol oxidase genes, Aox1 and Aox2, which have a strongly inducible promoter.[2] These genes allow Pichia to use methanol as a carbon and energy source. The AOX promoters are induced by methanol and are repressed by e.g. glucose. Usually, the gene for the desired protein is introduced under the control of the Aox1 promoter, which means that protein production can be induced by the addition of methanol. In a popular expression vector, the desired protein is produced as a fusion product to the secretion signal of the α-mating factor from Saccharomyces cerevisiae (baker's yeast). This causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. Some commercially available plasmids have these features incorporated (such as the pPICZα vector).[3]

Biotherapeutic production[edit]

Pichia pastoris was used for the production of over 500 biotherapeutics like interferon gamma. However, one drawback of this protein expression system is the over-glycosylation with high mannose structure which is a potential cause of immunogenicity.[4][5]

Comparison to other expression systems[edit]

In standard molecular biology research, the bacterium Escherichia coli is the most frequently used organism for production of recombinant DNA and proteins, due to E. coli's fast growth rate, good protein production rate, and undemanding growth conditions. Protein production in E. coli is usually faster than in P. pastoris for several reasons: Competent E. coli cells can be stored frozen, and thawed immediately before use, whereas Pichia cells have to be produced immediately before use. Expression yields in Pichia vary between different clones, and usually a large number of clones needs to be screened for protein production before a good producer is found. Optimal induction times of Pichia are usually on the order of days, whereas E. coli usually reaches optimal yields within hours of induction. The major advantage of Pichia over E. coli is that Pichia is capable of producing disulfide bonds and glycosylations in proteins.[6] This means that in cases where disulfides are necessary, E. coli might produce a misfolded protein, that is usually inactive or insoluble.[7]

The well-studied Saccharomyces cerevisiae is also used as an expression system with similar advantages over E. coli as Pichia. However Pichia has two main advantages over S. cerevisiae in laboratory and industrial settings:

Firstly, Pichia, as mentioned above, is a methylotroph, meaning it can grow with the simple alcohol methanol as its only source of energy — Pichia can easily be grown in cell suspension in reasonably strong methanol solutions that would kill most other micro-organisms, a system that is cheap to set up and maintain.

Secondly, Pichia can grow to very high cell densities, and under ideal conditions can multiply to the point where the cell suspension is practically a paste. As the protein yield from expression in a microbe is roughly equal to the product of the protein produced per cell and the number of cells, this makes Pichia of great use when trying to produce large quantities of protein without expensive equipment.[6]

Compared to other expression systems such as S2-cells from Drosophila melanogaster or Chinese hamster ovary cells, Pichia usually gives much better yields. Cell lines from multicellular organisms usually require complex rich media, including amino acids, vitamins, and growth factors. These media significantly increase the cost of producing recombinant proteins. Additionally, since Pichia can grow in media containing only one carbon source and one nitrogen source, it is suitable for isotopic labelling applications in e.g. protein NMR.[6] However, some proteins require chaperonins for proper folding. Thus, Pichia is unable to produce a number of proteins for which the host lacks the appropriate chaperones. In 2006, a research group managed to create a strain that produces erythropoietin in its normal glycosylation form.[8] This was achieved by exchanging the enzymes responsible for the fungal type glycosylation, with the mammalian homologs. Thus, the altered glycosylation pattern allowed the protein to be fully functional.

P. pastoris as a model organism[edit]

Another advantage of P. pastoris is its similarity to the well-studied Saccharomyces cerevisiae. As a model organism for biology, and having been used by man for various purposes throughout history, S. cerevisiae is well studied, to say the least. The two yeast species (Pichia, Saccharomyces) have similar growth conditions and tolerances; thus, the culture of P. pastoris can be readily adopted by labs without specialist equipment.

The P. pastoris GS115 genome has been sequenced by the Flanders Institute for Biotechnology and Ghent University, and published in Nature Biotechnology.[9] The genome sequence and gene annotation can be browsed through the ORCAE system.


  1. ^ Kurtzman CP (November 2009). "Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis". Journal of Industrial Microbiology & Biotechnology. 36 (11): 1435–8. doi:10.1007/s10295-009-0638-4. PMID 19760441.
  2. ^ Daly R, Hearn MT (2005). "Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production". Journal of Molecular Recognition. 18 (2): 119–38. doi:10.1002/jmr.687. PMID 15565717.
  3. ^ "Description of pPICZα vector by its vendor Invitrogen".
  4. ^ Razaghi A, Tan E, Lua LH, Owens L, Karthikeyan OP, Heimann K (January 2017). "Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma?". Biologicals. 45: 52–60. doi:10.1016/j.biologicals.2016.09.015. PMID 27810255.
  5. ^ Ali Razaghi; Roger Huerlimann; Leigh Owens; Kirsten Heimann (2015). "Increased expression and secretion of recombinant hIFNγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris". European Pharmaceutical Journal. 62 (2): 43–50. doi:10.1515/afpuc-2015-0031.
  6. ^ a b c Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T (2009). Expression in the yeast Pichia pastoris. Meth. Enzymol. Methods in Enzymology. 463. pp. 169–89. doi:10.1016/S0076-6879(09)63013-5. ISBN 978-0-12-374536-1. PMID 19892173.
  7. ^ Brondyk WH (2009). Selecting an appropriate method for expressing a recombinant protein. Meth. Enzymol. Methods in Enzymology. 463. pp. 131–47. doi:10.1016/S0076-6879(09)63011-1. ISBN 978-0-12-374536-1. PMID 19892171.
  8. ^ Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU (September 2006). "Humanization of yeast to produce complex terminally sialylated glycoproteins". Science. 313 (5792): 1441–3. doi:10.1126/science.1130256. PMID 16960007.
  9. ^ De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouzé P, Van de Peer Y, Callewaert N (June 2009). "Genome sequence of the recombinant protein production host Pichia pastoris". Nature Biotechnology. 27 (6): 561–6. doi:10.1038/nbt.1544. PMID 19465926.