''Pichia pastoris'' is a species of
methylotroph
Methylotrophs are a diverse group of microorganisms that can use reduced one-carbon compounds, such as methanol or methane, as the carbon source for their growth; and multi-carbon compounds that contain no carbon-carbon bonds, such as dimethyl e ...
ic
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
. It was found in the 1960s, with its feature of using
methanol
Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
as a source of carbon and energy.
After years of study, ''P. pastoris'' was widely used in
biochemical
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology an ...
research and
biotech industries. With strong potential for being an
expression system for
protein production
Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the tran ...
, as well as being a
model organism
A model organism (often shortened to model) is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workin ...
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
The Saccharomycetaceae are a family of yeasts in the order Saccharomycetales that reproduce by budding. Species in the family have a cosmopolitan distribution, and are present in a wide variety of habitats, especially those with a plentiful supp ...
'' with
phylogenetic analysis
In biology, phylogenetics (; from Greek φυλή/ φῦλον [] "tribe, clan, race", and wikt:γενετικός, γενετικός [] "origin, source, birth") is the study of the evolutionary history and relationships among or within groups o ...
, by
genome sequencing
Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...
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
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
,
glycerol
Glycerol (), also called glycerine in British English and glycerin in American English, is a simple triol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known ...
and
methanol
Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
.
However, they cannot use
lactose
Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix '' - ...
.
Reproduction
''P. pastoris'' can undergo both
asexual reproduction
Asexual reproduction is a type of reproduction that does not involve the fusion of gametes or change in the number of chromosomes. The offspring that arise by asexual reproduction from either unicellular or multicellular organisms inherit the fu ...
and
sexual
Sex is the biological distinction of an organism between male and female.
Sex or SEX may also refer to:
Biology and behaviour
*Animal sexual behaviour
**Copulation (zoology)
**Human sexual activity
**Non-penetrative sex, or sexual outercourse
** ...
reproduction, by
budding
Budding or blastogenesis is a type of asexual reproduction in which a new organism develops from an outgrowth or bud due to cell division at one particular site. For example, the small bulb-like projection coming out from the yeast cell is know ...
and
ascospore
An ascus (; ) is the sexual spore-bearing cell produced in ascomycete fungi. Each ascus usually contains eight ascospores (or octad), produced by meiosis followed, in most species, by a mitotic cell division. However, asci in some genera or s ...
.
In this case, two types of cells of ''P. pastoris'' exist:
haploid and diploid cells. In the asexual
life cycle
Life cycle, life-cycle, or lifecycle may refer to:
Science and academia
*Biological life cycle, the sequence of life stages that an organism undergoes from birth to reproduction ending with the production of the offspring
* Life-cycle hypothesis ...
, haploid cells undergo mitosis for reproduction. In the sexual life cycle, diploid cells undergo
sporulation
In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, f ...
and
meiosis
Meiosis (; , since it is a reductional division) is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately resu ...
.
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
ORCAEsystem. 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
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
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
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...
''. As a
model organism
A model organism (often shortened to model) is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workin ...
for biology, ''S. cerevisiae'' have been well studied for decades and used by researchers for various purposes throughout history. The two yeast genera; ''
Pichia
''Pichia'' (''Hansenula'' and ''Hyphopichia'' are obsolete synonyms) is a genus of yeasts in the family Pichiaceae with spherical, elliptical, or oblong acuminate cells. ''Pichia'' is a teleomorph, and forms hat-shaped, hemispherical, or round asc ...
'' and ''
Saccharomyces
''Saccharomyces'' is a genus of fungi that includes many species of yeasts. ''Saccharomyces'' is from Greek σάκχαρον (sugar) and μύκης (fungus) and means ''sugar fungus''. Many members of this genus are considered very important in f ...
'', 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
A peroxisome () is a membrane-bound organelle, a type of microbody, found in the cytoplasm of virtually all eukaryotic cells. Peroxisomes are oxidative organelles. Frequently, molecular oxygen serves as a co-substrate, from which hydrogen pero ...
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
The term heterologous has several meanings in biology.
Gene expression
In cell biology and protein biochemistry, heterologous expression means that a protein is experimentally put into a cell that does not normally make (i.e., express) that ...
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
Industrial fermentation is the intentional use of fermentation in manufacturing products useful to humans. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical industry. ...
, which makes it suitable for both small- and large-scale production.
2:''P. pastoris'' has two
alcohol oxidase
Alcohol most commonly refers to:
* Alcohol (chemistry), an organic compound in which a hydroxyl group is bound to a carbon atom
* Alcohol (drug), an intoxicant found in alcoholic drinks
Alcohol may also refer to:
Chemicals
* Ethanol, one of sev ...
genes, ''Aox1'' and ''Aox2'', which include strongly
inducible
Induction, Inducible or Inductive may refer to:
Biology and medicine
* Labor induction (birth/pregnancy)
* Induction chemotherapy, in medicine
* Induced stem cells, stem cells derived from somatic, reproductive, pluripotent or other cell typ ...
promoters.
These two genes allow ''Pichia'' to use methanol as a
carbon
Carbon () is a chemical element with the symbol C and atomic number 6. It is nonmetallic and tetravalent
In chemistry, the valence (US spelling) or valency (British spelling) of an element is the measure of its combining capacity with o ...
and energy source. The AOX promoters are induced by
methanol
Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
, and repressed by
glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
. 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
HSP60, also known as chaperonins (Cpn), is a family of heat shock proteins originally sorted by their 60kDa molecular mass. They prevent misfolding of proteins during stressful situations such as high heat, by assisting protein folding. HSP60 bel ...
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
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
research, the bacterium ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' is the most frequently used organism for expression system, to produce heterologous
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, 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
Clone or Clones or Cloning or Cloned or The Clone may refer to:
Places
* Clones, County Fermanagh
* Clones, County Monaghan, a town in Ireland
Biology
* Clone (B-cell), a lymphocyte clone, the massive presence of which may indicate a pathologi ...
, 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
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
bonds and
glycosylation
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
s 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
Methylotrophs are a diverse group of microorganisms that can use reduced one-carbon compounds, such as methanol or methane, as the carbon source for their growth; and multi-carbon compounds that contain no carbon-carbon bonds, such as dimethyl e ...
, meaning that it can grow with the simple methanol, as the only source of energy — ''Pichia'' can grow fast in
cell suspension
A cell suspension or suspension culture is a type of cell culture in which single Cell (biology), cells or small aggregates of cells are allowed to Cell physiology, function and Cell division, multiply in an agitated growth medium, thus forming a ...
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
''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...
'' and
Chinese hamster ovary cell
Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have foun ...
s, ''Pichia'' usually gives much better yields. Generally,
cell lines
An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
from multicellular organisms require complex and expensive types of media, including
amino acids
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
,
vitamin
A vitamin is an organic molecule (or a set of molecules closely related chemically, i.e. vitamers) that is an Nutrient#Essential nutrients, essential micronutrient that an organism needs in small quantities for the proper functioning of its ...
s, as well as other
growth factor
A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regu ...
s. 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
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
source, which is suitable for isotopic labelling applications, like
protein NMR Nuclear magnetic resonance spectroscopy of proteins (usually abbreviated protein NMR) is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and ...
.
Industrial applications
''P. pastoris'' have been used in several kinds of biotech industries, such as
pharmaceutical industry
The pharmaceutical industry discovers, develops, produces, and markets drugs or pharmaceutical drugs for use as medications to be administered to patients (or self-administered), with the aim to cure them, vaccinate them, or alleviate symptoms. ...
. 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
biotherapeutic
A biopharmaceutical, also known as a biological medical product, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semisynthesized from biological sources. Different from totally synthesized pharmaceuticals, t ...
s, such as
IFNγ
Interferon gamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferons. The existence of this interferon, which early in its history was known as immune interferon, was described by E. F. Wheelock ...
. At the beginning, one drawback of this protein expression system is the over-
glycosylation
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
with high density of
mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. It is a C-2 epimer of glucose. Mannose is important in human metabolism, especially in the glycosylation of certain proteins. Several congenital disorders of glycosylation ...
structure, which is a potential cause of
immunogenicity
Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted:
* Wanted immunogenicity typically relates to vaccines, where the injectio ...
.
In 2006, a research group managed to create a new strain called YSH597. This strain can express
erythropoietin
Erythropoietin (; EPO), also known as erythropoetin, haematopoietin, or haemopoietin, is a glycoprotein cytokine secreted mainly by the kidneys in response to cellular hypoxia; it stimulates red blood cell production (erythropoiesis) in the bo ...
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 aid A processing aid is a substance used in the production of processed food, and which may end up in the finished product, but which is not, by law, required to be disclosed to the consumer as an ingredient.
Ethical concerns
NGOs, journalists, and f ...
s and
food additive
Food additives are substances added to food to preserve flavor or enhance taste, appearance, or other sensory qualities. Some additives have been used for centuries as part of an effort to preserve food, for example vinegar (pickling), salt (salt ...
s, 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