Pichia pastoris

Pichia pastoris
Scientific classification
Kingdom:
Fungi
Phylum:
Ascomycota
Class:
Saccharomycetes
Order:
Saccharomycetales
Family:
Saccharomycetaceae
Genus:
Pichia / Komagataella
Species:
pastoris
Binomial name
Pichia pastoris

Pichia pastoris is a species of methylotrophic yeast. It was found in the 1960s, with its feature of using methanol as a source of carbon and energy.[1] After years of study, P. pastoris was widely used in biochemical research and biotech industries. With strong potential for being an expression system for protein production, as well as being a model organism for genetic study, P. pastoris has become important for biological research and biotech applications. In the last decade, some reports reassigned P. pastoris to the genus Komagataella with phylogenetic analysis, by genome sequencing of P. pastoris. The genus was split into K. phaffii, K. pastoris, and K. pseudopastoris.[2][3]

P. pastoris in nature

Natural habitat

Naturally, P. pastoris lives on trees, such as chestnut trees. They are heterotrophs and they can use several carbon sources for living, like glucose, glycerol and methanol.[4] However, they cannot use lactose.

Reproduction

P. pastoris can undergo both asexual reproduction and sexual reproduction, by budding and ascospore.[5] In this case, two types of cells of P. pastoris exist: haploid and diploid cells. In the asexual life cycle, haploid cells undergo mitosis for reproduction. In the sexual life cycle, diploid cells undergo sporulation and meiosis.[6] The growth rate of its colonies can vary by a large range, from near to 0 to a doubling time of one hour, which is suitable for industrial processes.[7]

P. pastoris as a model organism

In the last few years, P. pastoris was investigated and identified as a good model organism with several advantages. First of all, P. patoris can be grown and used easily in lab. Like other widely used yeast models, it has relatively short life span and fast regeneration time. Moreover, some inexpensive culture media have been designed, so that P. patoris can grow quickly on them, with high cell density.[8] Whole genome sequencing for P. patoris had been performed. 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. The complete genomic data allows scientists to identify homologous proteins and evolutionary relationships between other yeast species and P. pastoris. Furthermore, P. pastoris are single eukaryotic cells, which means researchers could investigate the proteins inside P. pastoris. Then the homologous comparison to other more complicated eukaryotic species can be processed, to obtain their functions and origins.[10]

Another advantage of P. pastoris is its similarity to the well-studied yeast model — Saccharomyces cerevisiae. As a model organism for biology, S. cerevisiae have been well studied for decades and used by researchers for various purposes throughout history. The two yeast genera; Pichia and Saccharomyces, have similar growth conditions and tolerances; thus, the culture of P. pastoris can be adopted by labs without many modifications.[11] Moreover, unlike S. cerevisiae, P. pastoris has the ability to functionally process proteins with large molecular weight, which is useful in a translational host.[12] Considering all the advantages, P. pastoris can be usefully employed as both a genetic and experimental model organism.

P. pastoris as a genetic model organism

As a genetic model organism, P. pastoris can be used for genetic analysis and large-scale genetic crossing, with complete genome data and its ability to carry out complex eukaryotic genetic processing in a relatively small genome. The functional genes for peroxisome assembly were investigated by comparing wild-type and mutant strains of P. pastoris.[13]

P. pastoris as an experimental model organism

As an experimental model organism, P. pastoris was mainly used as the host system for transformation. Due to its abilities of recombination with foreign DNA and processing large proteins, much research has been carried out to investigate the possibility of producing new proteins and the function of artificially designed proteins, using P. pastoris as a transformation host.[14] In the last decade, P. pastoris was engineered to build expression system platforms, which is a typical application for a standard experimental model organism, as described below.

P. pastoris as expression system platform

P. pastoris is frequently used as an expression system for the production of heterologous proteins. Several properties make P. pastoris suited for this task. Currently, several strains of P. pastoris are used for biotechnical purposes, with significant differences among them in growth and protein production.[15] Some common variants possess a mutation in the HIS4 gene, leading to the selection of cells which are transformed successfully with expression vectors. The technology for vector integration into P. pastoris genome is similar to that in Saccharomyces cerevisiae.[16]

Advantage

1:P. pastoris is able to grow on simple, inexpensive medium, with high growth rate. P. pastoris can grow in either shake flasks or a fermenter, which makes it suitable for both small- and large-scale production.[17]

2:P. pastoris has two alcohol oxidase genes, Aox1 and Aox2, which include strongly inducible promoters.[18] These two genes allow Pichia to use methanol as a carbon and energy source. The AOX promoters are induced by methanol, and repressed by 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 on medium. After several researches, scientists found that the promotor derived from AOX1 gene in P. pastoris is extremely suitable to control the expression of foreign genes, which had been transformed into the P. pastoris genome, producing heterologous proteins.[19]

3: With a key trait, P. pastoris can grow with extremely high cell density on the culture. This feature is compatible with heterologous protein expression, giving higher yields of production.[20]

4: The technology required for genetic manipulation of P. pastoris is similar to that of Saccharomyces cerevisiae, which is one of the most well-studied yeast model organisms. As a result, the experiment protocol and materials are easy to build for P. pastoris.[21]

Disadvantage

As some proteins require chaperonin for proper folding, Pichia is unable to produce a number of proteins, since P. pastoris does not contain the appropriate chaperones. The technologies of introducing genes of mammalian chaperonins into the yeast genome and overexpressing existing chaperonins still require improvement.[22][23]

Comparison with other expression systems

In standard molecular biology research, the bacterium Escherichia coli is the most frequently used organism for expression system, to produce heterologous proteins, due to its features of fast growth rate, high protein production rate, as well as undemanding growth conditions. Protein production in E. coli is usually faster than that in P. pastoris, with reasons: Competent E. coli cells can be stored frozen, and thawed before use, whereas Pichia cells have to be produced immediately before use. Expression yields in Pichia vary between different clones, so that a large number of clones has to be screened for protein production, to find the best producer. The biggest advantage of Pichia over E. coli is that Pichia is capable of forming disulfide bonds and glycosylations in proteins, but E. coli cannot.[24] E. coli might produce a misfolded protein when disulfides are included in final product, leading to inactive or insoluble forms of proteins.[25]

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:

  1. Pichia, as mentioned above, is a methylotroph, meaning that it can grow with the simple methanol, as the only source of energy — Pichia can grow fast in cell suspension with reasonably strong methanol solution, which would kill most other micro-organisms. In this case, the expression system is cheap to set up and maintain.
  2. Pichia can grow up to a very high cell density. Under ideal conditions, it can multiply to the point where the cell suspension is practically a paste. As the protein yield from expression system in a microbe is roughly equal to the product of the proteins produced per cell, which makes Pichia of great use when trying to produce large quantities of protein without expensive equipment.[24]

Comparing to other expression systems, such as S2-cells from Drosophila melanogaster and Chinese hamster ovary cells, Pichia usually gives much better yields. Generally, cell lines from multicellular organisms require complex and expensive types of media, including amino acids, vitamins, as well as other growth factors. These types of media significantly increase the cost of producing heterologous proteins. Additionally, since Pichia can grow in media containing only one carbon source and one nitrogen source, which is suitable for isotopic labelling applications, like protein NMR.[24]

Industrial applications

P. pastoris have been used in several kinds of biotech industries, such as pharmaceutical industry. All the applications are based on its feature of expressing proteins.

Biotherapeutic production

In the last few years, Pichia pastoris had been used for the production of over 500 types of biotherapeutics, such as IFNγ. At the beginning, one drawback of this protein expression system is the over-glycosylation with high density of mannose structure, which is a potential cause of immunogenicity.[26][27] In 2006, a research group managed to create a new strain called YSH597. This strain can express erythropoietin in its normal glycosylation form, 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.[28]

Enzyme production for food industry

In food industries, like brewery and bake house, Pichia pastoris is used to produce different kinds of enzymes, as processing aids and food additives, with many functions. For example, some enzymes produced by genetically modified Pichia pastoris can keep the bread soft. Meanwhile, in beer, enzymes could be used to lower the alcohol concentration.[29]

References

  1. ↑ Koichi Ogata, Hideo Nishikawa & Masahiro Ohsugi (1969). "A Yeast Capable of Utilizing Methanol". Agricultural and Biological Chemistry. 33 (10): 1519–1520. doi:10.1080/00021369.1969.10859497.
  2. ↑ De Schutter, K., Lin, Y., Tiels, P. (2009). "Genome sequence of the recombinant protein production host Pichia pastoris". Nature Biotechnology. 27 (6): 561–566. doi:10.1038/nbt.1544. PMID 19465926.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. ↑ Heistinger, Lina; Gasser, Brigitte; Mattanovich, Diethard (2020-07-01). "Microbe Profile: Komagataella phaffii: a methanol devouring biotech yeast formerly known as Pichia pastoris". Microbiology. 166 (7): 614–616. doi:10.1099/mic.0.000958. ISSN 1350-0872. PMID 32720891.
  4. ↑ Rebnegger, C., Vos, T., Graf, A. B., Valli, M., Pronk, J. T., Daran-Lapujade, P., & Mattanovich, D. (2016). "Pichia pastoris exhibits high viability and a low maintenance energy requirement at near-zero specific growth rates". Applied and Environmental Microbiology. 82 (15): 4570–4583. Bibcode:2016ApEnM..82.4570R. doi:10.1128/AEM.00638-16. PMC 4984280. PMID 27208115.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ↑ Kurtzman (1998). "42 - Pichia E.C. Hansen emend. Kurtzman". The Yeasts: A Taxonomic Study. 1: 273–352. doi:10.1016/B978-044481312-1/50046-0. ISBN 9780444813121.
  6. ↑ Zörgö E, Chwialkowska K, Gjuvsland AB, Garré E, Sunnerhagen P, Liti G, Blomberg A, Omholt SW, Warringer J (2013). "Ancient Evolutionary Trade-Offs between Yeast Ploidy States". PLOS Genetics. 9 (3): e1003388. doi:10.1371/journal.pgen.1003388. PMC 3605057. PMID 23555297.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  7. ↑ Kastilan, R., Boes, A., Spiegel, H. (2017). "Improvement of a fermentation process for the production of two PfAMA1-DiCo-based malaria vaccine candidates in Pichia pastoris". Nature. 1 (1): 7. Bibcode:2017NatSR...711991K. doi:10.1038/s41598-017-11819-4. PMC 5607246. PMID 28931852.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ↑ M. M. Guarna G. J. Lesnicki B. M. Tam J. Robinson C. Z. Radziminski D. Hasenwinkle A. Boraston E. Jervis R. T. A. MacGillivray R. F. B. Turner D. G. Kilburn (1997). "On‐line monitoring and control of methanol concentration in shake‐flask cultures of Pichia pastoris". Biotechnology and Bioengineering. 56 (3): 279–286. doi:10.1002/(SICI)1097-0290(19971105)56:3<279::AID-BIT5>3.0.CO;2-G. PMID 18636643.
  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.
  10. ↑ Brigitte Gasser, Roland Prielhofer, Hans Marx, Michael Maurer, Justyna Nocon, Matthias Steiger, Verena Puxbaum, Michael Sauer & Diethard Mattanovich (2013). "Pichia pastoris: protein production host and model organism for biomedical research". Future Microbiology. 8 (2): 191–208. doi:10.2217/fmb.12.133. PMID 23374125.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  11. ↑ Tran, A., Nguyen, T., Nguyen, C. (2017). "Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor". BMC Res Notes. 10 (1): 148. doi:10.1186/s13104-017-2471-6. PMC 5379694. PMID 28376863.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  12. ↑ Heidebrecht, Aniela, and Thomas Scheibel (2013). "Recombinant production of spider silk proteins". Advances in Applied Microbiology. 82: 115–153. doi:10.1016/B978-0-12-407679-2.00004-1. ISBN 9780124076792. PMID 23415154.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. ↑ Gould, S. J., McCollum, D., Spong, A. P., Heyman, J. A., & Subramani, S. (1992). "Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly". The Yeasts: A Taxonomic Study. 8 (8): 613–628. doi:10.1002/yea.320080805. PMID 1441741. S2CID 8840145.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  14. ↑ Cregg, J. M., Barringer, K. J., Hessler, A. Y., & Madden, K. R. (1985). "Pichia pastoris as a host system for transformations". Molecular and Cellular Biology. 5 (12): 3376–3385. doi:10.1128/MCB.5.12.3376. PMC 369166. PMID 3915774.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ↑ Brady, J.R. (2020). "Comparative genome-scale analysis of Pichia pastoris variants informs selection of an optimal base strain". Biotechnology and Bioengineering. 117 (2): 543–555. doi:10.1002/bit.27209. PMC 7003935. PMID 31654411.
  16. ↑ Higgins, D. R., & Cregg, J. M. (1998). "Introduction to Pichia pastoris". Pichia Protocols. 103: 1–15. doi:10.1385/0-89603-421-6:1. ISBN 0-89603-421-6. PMID 9680629.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ↑ Wenhui Zhang Mark A. Bevins Bradley A. Plantz Leonard A. Smith Michael M. Meagher. (2000). "Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy‐chain fragment C of botulinum neurotoxin, serotype A". Biotechnology and Bioengineering. 70 (1): 1–8. doi:10.1002/1097-0290(20001005)70:1<1::AID-BIT1>3.0.CO;2-Y. PMID 10940857.
  18. ↑ 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. S2CID 7476149.
  19. ↑ Romanos, Mike. (1995). "Advances in the use of Pichia pastoris for high-level gene expression". Current Opinion in Biotechnology. 6 (5): 527–533. doi:10.1016/0958-1669(95)80087-5.
  20. ↑ Zhou, X., Yu, Y., Tao, J., & Yu, L. (2014). "Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation". Journal of Bioscience and Bioengineering . 118 (4): 420–425. doi:10.1016/j.jbiosc.2014.03.009. PMID 24745549.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  21. ↑ Morton, C. L., & Potter, P. M. (2000). "Comparison of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda, and COS7 cells for recombinant gene expression". Molecular Biotechnology. 16 (3): 193–202. doi:10.1385/MB:16:3:193. PMID 11252804. S2CID 22792748.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  22. ↑ Bankefa, OE; Wang, M; Zhu, T; Li, Y (July 2018). "Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris". Biotechnology Letters. 40 (7): 1149–1156. doi:10.1007/s10529-018-2571-y. PMID 29785668. S2CID 29155989.
  23. ↑ Yu, Xiao-Wei; Sun, Wei-Hong; Wang, Ying-Zheng; Xu, Yan (24 November 2017). "Identification of novel factors enhancing recombinant protein production in multi-copy Komagataella phaffii based on transcriptomic analysis of overexpression effects". Scientific Reports. 7 (1): 16249. Bibcode:2017NatSR...716249Y. doi:10.1038/s41598-017-16577-x. PMC 5701153. PMID 29176680.
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  25. ↑ Brondyk WH (2009). Selecting an appropriate method for expressing a recombinant protein. Meth. Enzymol. Methods in Enzymology. Vol. 463. pp. 131–47. doi:10.1016/S0076-6879(09)63011-1. ISBN 978-0-12-374536-1. PMID 19892171.
  26. ↑ 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.
  27. ↑ 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.
  28. ↑ 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. Bibcode:2006Sci...313.1441H. doi:10.1126/science.1130256. PMID 16960007. S2CID 43334198.
  29. ↑ Spohner, S. C., Müller, H., Quitmann, H., & Czermak, P. (2015). "Expression of enzymes for the usage in food and feed industry with Pichia pastoris". Journal of Biotechnology. 202: 420–425. doi:10.1016/j.jbiotec.2015.01.027. PMID 25687104.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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