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. 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''.

''P. pastoris'' in nature

Natural habitat

In nature, ''P. pastoris'' is found on trees, such as chestnut trees. They are heterotrophs and they can use several carbon sources for living, like glucose, glycerol and methanol. However, they cannot use lactose.

Reproduction

''P. pastoris'' can undergo both asexual reproduction and sexual reproduction, by budding and ascospore. 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. 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.

''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. pastoris'' 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. pastoris'' can grow quickly on them, with high cell density.
Whole genome sequencing for ''P. pastoris'' 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''. The genome sequence and gene annotation can be browsed through th
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.

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. Moreover, unlike ''S. cerevisiae'', ''P. pastoris'' has the ability to functionally process proteins with large molecular weight, which is useful in a translational host.
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''.

''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. 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. 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''.

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.

2:''P. pastoris'' has two alcohol oxidase genes, ''Aox1'' and ''Aox2'', which include strongly inducible promoters. 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.

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.

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''.

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.

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. ''E. coli'' might produce a misfolded protein when disulfides are included in final product, leading to inactive or insoluble forms of proteins.

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:
#''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.
#''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.

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.

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. 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.

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.

References

{{Taxonbar, from=Q148697

Saccharomycetaceae
Fungal models
Yeasts
Fungi described in 1956