Pichia pastoris

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Komagataella
Pichia pastoris GS115 (Yang 2017).jpg
Komagataella phaffii [1] GS115
Scientific classification OOjs UI icon edit-ltr.svg
Domain: Eukaryota
Kingdom: Fungi
Division: Ascomycota
Class: Saccharomycetes
Order: Saccharomycetales
Family: Phaffomycetaceae
Genus: Komagataella
Y. Yamada, M. Matsuda, K. Maeda & Mikata, 1995
Species

See text

Komagataella is a methylotrophic yeast within the order Saccharomycetales. It was found in the 1960s as Pichia pastoris, with its feature of using methanol as a source of carbon and energy. [2] In 1995, P. pastoris was reassigned into the sole representative of genus Komagataella, becoming Komagataella pastoris. [3] Later studies have further distinguished new species in this genus, resulting in a total of 7 recognized species. [4] It is not uncommon to see the old name still in use, as of 2023; in less formal use, the yeast may confusingly be referred to as pichia.

Contents

After years of study, Komagataella is 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, Komagataella phaffii has become important for biological research and biotech applications. [1] [5]

Taxonomy

According to GBIF: [4]

Komagataella in nature

Natural habitat

In nature, Komagataella is found on trees, such as chestnut trees. [6] They are heterotrophs and they can use several carbon sources for living, like glucose, glycerol and methanol. [7] However, they cannot use lactose.

Reproduction

Komagataella can undergo both asexual reproduction and sexual reproduction, by budding and ascospore. [8] In this case, two types of cells of Komagataella 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. [9] 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. [10]

Komagataella as a model organism

In the last few years, Komagataella was investigated and identified as a good model organism with several advantages. First of all, Komagataella 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 Komagataella can grow quickly on them, with high cell density. [11] Whole genome sequencing for Komagataella had been performed. The K. phaffii GS115 genome has been sequenced by the Flanders Institute for Biotechnology and Ghent University, and published in Nature Biotechnology. [12] 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 Komagataella. In addition, all seven species were sequenced by 2022. [6] Furthermore, Komagataella are single eukaryotic cells, which means researchers could investigate the proteins inside Komagataella. Then the homologous comparison to other more complicated eukaryotic species can be processed, to obtain their functions and origins. [13]

Another advantage of Komagataella 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 (sensu lato) and Saccharomyces , have similar growth conditions and tolerances; thus, the culture of Komagataella can be adopted by labs without many modifications. [14] Moreover, unlike S. cerevisiae, Komagataella has the ability to functionally process proteins with large molecular weight, which is useful in a translational host. [15] Considering all the advantages, Komagataella can be usefully employed as both a genetic and experimental model organism.

Komagataella as a genetic model organism

As a genetic model organism, Komagataella 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 Komagataella. [16]

Komagataella as an experimental model organism

As an experimental model organism, Komagataella 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 Komagataella as a transformation host. [17] In the last decade, Komagataella was engineered to build expression system platforms, which is a typical application for a standard experimental model organism, as described below.

Komagataella as expression system platform

Komagataella is frequently used as an expression system for the production of heterologous proteins. Several properties make Komagataella suited for this task. Currently, several strains of Komagataella are used for biotechnical purposes, with significant differences among them in growth and protein production. [18] 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 Komagataella genome is similar to that in Saccharomyces cerevisiae. [19]

Advantage

  1. Komagataella is able to grow on simple, inexpensive medium, with high growth rate. Komagataella can grow in either shake flasks or a fermenter, which makes it suitable for both small- and large-scale production. [20]
  2. Komagataella has two alcohol oxidase genes, Aox1 and Aox2, which include strongly inducible promoters. [21] These two genes allow Komagataella 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 Komagataella is extremely suitable to control the expression of foreign genes, which had been transformed into the Komagataella genome, producing heterologous proteins. [22]
  3. With a key trait, Komagataella can grow with extremely high cell density on the culture. This feature is compatible with heterologous protein expression, giving higher yields of production. [23]
  4. The technology required for genetic manipulation of Komagataella 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 Komagataella. [24]

Disadvantage

As some proteins require chaperonin for proper folding, Komagataella is unable to produce a number of proteins, since it 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. [25] [26]

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 Komagataella, with reasons: Competent E. coli cells can be stored frozen, and thawed before use, whereas Komagataella cells have to be produced immediately before use. Expression yields in Komagataella 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 Komagataella over E. coli is that Komagataella is capable of forming disulfide bonds and glycosylations in proteins, but E. coli cannot. [27] E. coli might produce a misfolded protein when disulfides are included in final product, leading to inactive or insoluble forms of proteins. [28]

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

  1. Komagataella, as mentioned above, is a methylotroph, meaning that it can grow with the simple methanol, as the only source of energy — Komagataella 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. Komagataella 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 Komagataella of great use when trying to produce large quantities of protein without expensive equipment. [27]

Comparing to other expression systems, such as S2-cells from Drosophila melanogaster and Chinese hamster ovary cells, Komagataella 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, Komagataella can grow in media containing only one carbon source and one nitrogen source, which is suitable for isotopic labelling applications, like protein NMR. [27]

Industrial applications

Komagataella 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, Komagataella 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. [29] [30] In 2006, a research group managed to create a new strain called YSH597. [lower-alpha 1] 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. [31]

Enzyme production

In food industries, like brewery and bake house, Komagataella 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 Komagataella can keep the bread soft. Meanwhile, in beer, enzymes could be used to lower the alcohol concentration. [32] Recombinant phospholipase C can degum high-phosphorus oils by breaking down phospholipids. [33]

In animal feed, K. phaffi-produced phytase is used to break down phytic acid, an antinutrient. [33]

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References

  1. ↑ YSH597 is based on strain NRRL-Y11430, now considered part of K. phaffi.
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  28. ↑ Brondyk WH (2009). "Chapter 11 Selecting an Appropriate Method for Expressing a Recombinant Protein". Guide to Protein Purification, 2nd Edition. pp. 131–47. doi:10.1016/S0076-6879(09)63011-1. ISBN   978-0-12-374536-1. PMID   19892171.{{cite book}}: |journal= ignored (help)
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  32. ↑ 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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