Komagataella

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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 phaffii. [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 in the context of protein production, as of 2023; [5] 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. [7] They are heterotrophs and they can use several carbon sources for living, like glucose, glycerol and methanol. [8] However, they cannot use lactose.

Reproduction

Komagataella can undergo both asexual reproduction and sexual reproduction, by budding and ascospore. [9] 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. [10] 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. [11]

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. [12] 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. [13] 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. [7] 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. [14]

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. [15] Moreover, unlike S. cerevisiae, Komagataella has the ability to functionally process proteins with large molecular weight, which is useful in a translational host. [16] 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. [17]

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. [18] 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. [19] 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. [20]

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. [21]
  2. Komagataella has two alcohol oxidase genes, Aox1 and Aox2, which include strongly inducible promoters. [22] 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. [23]
  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. [24]
  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. [25]

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. [26] [27]

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. [28] E. coli might produce a misfolded protein when disulfides are included in final product, leading to inactive or insoluble forms of proteins. [29]

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. [28]

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. [28]

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. [30] [31] 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. [32]

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. [33] Recombinant phospholipase C can degum high-phosphorus oils by breaking down phospholipids. [34]

In animal feed, K. phaffi-produced phytase is used to break down phytic acid, an antinutrient. [34]

Related Research Articles

<span class="mw-page-title-main">Protein production</span>

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 transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

<span class="mw-page-title-main">Codon usage bias</span> Genetic bias in coding DNA

Codon usage bias refers to differences in the frequency of occurrence of synonymous codons in coding DNA. A codon is a series of three nucleotides that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation.

<i>Saccharomyces cerevisiae</i> Species of yeast

Saccharomyces cerevisiae is a species of yeast. The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been originally isolated from the skin of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism which causes many common types of fermentation. S. cerevisiae cells are round to ovoid, 5–10 μm in diameter. It reproduces by budding.

<span class="mw-page-title-main">Yeast artificial chromosome</span> Genetically engineered chromosome derived from the DNA of yeast

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

The word metagenics uses the prefix meta and the suffix gen. Literally, it means "the creation of something which creates". In the context of biotechnology, metagenics is the practice of engineering organisms to create a specific enzyme, protein, or other biochemicals from simpler starting materials. The genetic engineering of E. coli with the specific task of producing human insulin from starting amino acids is an example. E. coli has also been engineered to digest plant biomass and use it to produce hydrocarbons in order to synthesize biofuels. The applications of metagenics on E. coli also include higher alcohols, fatty-acid based chemicals and terpenes.

Recombineering is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.

<i>Pichia</i> Genus of fungi

Pichia 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 ascospores during sexual reproduction. The anamorphs of some Pichia species are Candida species. The asexual reproduction is by multilateral budding.

Fed-batch culture is, in the broadest sense, defined as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. An alternative description of the method is that of a culture in which "a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion". It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor. The advantage of the fed-batch culture is that one can control concentration of fed-substrate in the culture liquid at arbitrarily desired levels.

Cell wall protein 2 (CWP2) is a cell wall protein, produced by Saccharomyces cerevisiae and Saccharomyces pastorianus. It occurs throughout the cell wall and has close homology with the CWP1 gene.

<i>Ogataea polymorpha</i> Species of fungus

Ogataea polymorpha is a methylotrophic yeast with unusual characteristics. It is used as a protein factory for pharmaceuticals.

A yeast expression platform is a strain of yeast used to produce large amounts of proteins, sugars or other compounds for research or industrial uses. While yeast are often more resource-intensive to maintain than bacteria, certain products can only be produced by eukaryotic cells like yeast, necessitating use of a yeast expression platform. Yeasts differ in productivity and with respect to their capabilities to secrete, process and modify proteins. As such, different types of yeast are better suited for different research and industrial applications.

<span class="mw-page-title-main">Xylose metabolism</span>

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A subunit vaccine is a vaccine that contains purified parts of the pathogen that are antigenic, or necessary to elicit a protective immune response. Subunit vaccine can be made from dissembled viral particles in cell culture or recombinant DNA expression, in which case it is a recombinant subunit vaccine.

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Heterologous expression refers to the expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment in question. Insertion of the gene in the heterologous host is performed by recombinant DNA technology. The purpose of heterologous expression is often to determine the effects of mutations and differential interactions on protein function. It provides an easy path to efficiently express and experiment with combinations of genes and mutants that do not naturally occur.

Aerobic fermentation or aerobic glycolysis is a metabolic process by which cells metabolize sugars via fermentation in the presence of oxygen and occurs through the repression of normal respiratory metabolism. Preference of aerobic fermentation over aerobic respiration is referred to as the Crabtree effect in yeast, and is part of the Warburg effect in tumor cells. While aerobic fermentation does not produce adenosine triphosphate (ATP) in high yield, it allows proliferating cells to convert nutrients such as glucose and glutamine more efficiently into biomass by avoiding unnecessary catabolic oxidation of such nutrients into carbon dioxide, preserving carbon-carbon bonds and promoting anabolism.

References

  1. ↑ YSH597 is based on strain NRRL-Y11430, now considered part of K. phaffi.
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