Hansenula polymorpha
Encyclopedia
Ogataea polymorpha is a methylotrophic yeast
with unusual characteristics. It is used as a protein factory for pharmaceuticals.
Ogataea polymorpha belongs to a limited number of methylotrophic yeast species – yeasts that can grow on methanol
. The range of methylotrophic yeasts includes Candida boidinii, Pichia methanolica, Pichia pastoris
and Ogataea polymorpha. O. polymorpha is taxonomically a species of the Saccharomycetaceae
family.
Strains CBS4732 and NCYY495 can be mated whereas strain DL-1 cannot be mated with the other two. Strains CBS4732 and DL-1 are employed for recombinant protein production, strain NCYC495 is mainly used for the study of nitrate assimilation.
The entire genome
of strain CBS4732 has completely been sequenced.
O. polymorpha is a thermo-tolerant microorganism with some strains growing at temperatures above 50 °C (122 °F). The organism is able to assimilate nitrate
and can grow on a range of carbon sources in addition to methanol
. Cells grown under conditions of elevated temperature accumulate a sugar named trehalose
(this sugar is usually found in insects) as thermo-protective compound. It was shown that trehalose synthesis is not required for growth under these conditions, but for acquisition of thermotolerance. The synthetic steps for trehalose synthesis have been detailed for O. polymorpha, and TPS1, the key enzyme gene of this pathway, has been isolated and characterized.
All methylotrophic yeasts share an identical methanol utilization pathway (Fig. 1) Growth on methanol is accompanied by a massive proliferation of cell organelle
s named peroxisomes in which the initial enzymatic steps of this pathway take place. O. polymorpha is model organism to study all aspects of peroxisomal functions and the underlying molecular biology. During growth on methanol key enzymes of the methanol metabolism are present in high amounts. An especially high abundance can be observed for enzymes called MOX (methanol oxidase), FMDH (formate dehydrogenase), and DHAS (dihydroxyacetone synthase). Their presence is regulated at the transcriptional level of the respective genes. In the related species C. boidinii, P. methanolica, and P. pastoris this gene expression strictly depends on the presence of methanol or methanol derivatives, whereas in O. polymorpha strong expression is elicited by appropriate levels of glycerol or under conditions of glucose starvation. O. polymorpha produces glycoproteins with two types of sugar chains, N- and O-linked glycans are attached to protein. Studies on the structure of N-linked chains have revealed a certain average length (Man8-12GlcNAc2) with terminal alpha-1,2-linked mannose residues, and not with allergenic terminal alpha-1,3-linked mannose residues as found in other yeasts, especially in the baker’s yeast Saccharomyces cerevisiae.
for treatment of diabetes, hepatitis B vaccines or IFNalpha-2a for the treatment of hepatitis C
. Derivatives of both CBS4732 and DL-1 are employed in the production of such recombinant compounds. Further yeasts employed for this application are Pichia pastoris, Arxula adeninivorans
and Saccharomyces cerevisiae
and others.
Like other yeasts, O. polymorpha is a microorganism that can be cultured in large fermenters to high cell densities within a short time. It is a safe organ-ism in not containing pyrogens, pathogens or viral inclusions. It can release compounds into a culture medium as it has all the components required for secretion (this is for instance not the case with bacteria like Escherichia coli
). It can provide attractive genetic components for an efficient production of proteins.
In Fig. 2 the general design of a vector (a genetic vehicle to transform a yeast strain into a genetically engineered protein producer). It must contain several genetic elements:
1. A selection marker, required to select a transformed strain from an untransformed background –this can be done if for instance such an element enables a deficient strain to grow under culturing conditions void of a certain compound like a particular amino acid that cannot be produced by the deficient strain).
2. Certain elements to propagate and to target the foreign DNA to the chromosome of the yeast (ARS and/or rDNA sequence).
3. A segment responsible for the production of the desired protein compound a so-called expression cassette. Such a cassette is made up by a sequence of regulatory elements, a promoter that controls, how much and under which circumstances a following gene sequence is transcribed and as a consequence how much protein is eventually made. This means that the segment following the promoter is variable depending on the desired product – it could be a sequence determining the amino acids for insulin, for hepatitis B vaccine or for interferon. The expression cassette is terminated by a following terminator sequence that provides a proper stop of the transcription. The promoter elements of the O. polymorpha system are derived from genes that are highly expressed, from instance from the MOX gene, the FMD gene or the TPS1 gene mentioned before. They are not only very strong, but can also be regulated by certain addition of carbon sources like sugar, methanol or glycerol.
In 2000 an informal society of scientists was founded named HPWN (Hansenula polymorpha worldwide network) founded by Prof. Marten Veenhuis, Groningen, and Prof, Dr. Gerd Gellissen, Düsseldorf. Every two years meetings are organized.
The attractiveness of the O. polymorpha platform is commercially exploited by several biotech companies for the development of production processes, among others by PharmedArtis, located in Aachen, Germany and the Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK).
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
with unusual characteristics. It is used as a protein factory for pharmaceuticals.
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
. The range of methylotrophic yeasts includes Candida boidinii, Pichia methanolica, Pichia pastoris
Pichia pastoris
Pichia pastoris is a species of methylotrophic yeast. Pichia is widely used for protein expression using recombinant DNA techniques. Hence it is used in biochemical and genetic research in academia and the biotechnical industry....
and Ogataea polymorpha. O. polymorpha is taxonomically a species of the Saccharomycetaceae
Saccharomycetaceae
The Saccharomycetaceae are a family of yeast in the order Saccharomycetales that reproduce by budding. Species in the family have a cosmopolitan distribution distribution, and are present in a wide variety of habitats, especially those with a plentiful supply of carbohydrate sources...
family.
Strains
Three basic O. polymorpha strains with unclear relationships and of independent origins exist identified in the fifties in soil samples, the gut of insects or in spoiled concentrated orange juice. They exhibit different features and are applied to basic research and to recombinant protein production:- strain CBS4732 (CCY38-22-2; ATCC34438, NRRL-Y-5445)
- strain DL-1 (NRRL-Y-7560; ATCC26012)
- strain NCYC495 (CBS1976; ATAA14754, NRLL-Y-1798)
Strains CBS4732 and NCYY495 can be mated whereas strain DL-1 cannot be mated with the other two. Strains CBS4732 and DL-1 are employed for recombinant protein production, strain NCYC495 is mainly used for the study of nitrate assimilation.
The entire genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of strain CBS4732 has completely been sequenced.
O. polymorpha is a thermo-tolerant microorganism with some strains growing at temperatures above 50 °C (122 °F). The organism is able to assimilate nitrate
Nitrate
The nitrate ion is a polyatomic ion with the molecular formula NO and a molecular mass of 62.0049 g/mol. It is the conjugate base of nitric acid, consisting of one central nitrogen atom surrounded by three identically-bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a...
and can grow on a range of carbon sources in addition to methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
. Cells grown under conditions of elevated temperature accumulate a sugar named trehalose
Trehalose
Trehalose, also known as mycose or tremalose, is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. In 1832, H.A.L. Wiggers discovered trehalose in an ergot of rye, and in 1859 Marcellin Berthelot isolated it from trehala manna, a substance made...
(this sugar is usually found in insects) as thermo-protective compound. It was shown that trehalose synthesis is not required for growth under these conditions, but for acquisition of thermotolerance. The synthetic steps for trehalose synthesis have been detailed for O. polymorpha, and TPS1, the key enzyme gene of this pathway, has been isolated and characterized.
All methylotrophic yeasts share an identical methanol utilization pathway (Fig. 1) Growth on methanol is accompanied by a massive proliferation of cell organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....
s named peroxisomes in which the initial enzymatic steps of this pathway take place. O. polymorpha is model organism to study all aspects of peroxisomal functions and the underlying molecular biology. During growth on methanol key enzymes of the methanol metabolism are present in high amounts. An especially high abundance can be observed for enzymes called MOX (methanol oxidase), FMDH (formate dehydrogenase), and DHAS (dihydroxyacetone synthase). Their presence is regulated at the transcriptional level of the respective genes. In the related species C. boidinii, P. methanolica, and P. pastoris this gene expression strictly depends on the presence of methanol or methanol derivatives, whereas in O. polymorpha strong expression is elicited by appropriate levels of glycerol or under conditions of glucose starvation. O. polymorpha produces glycoproteins with two types of sugar chains, N- and O-linked glycans are attached to protein. Studies on the structure of N-linked chains have revealed a certain average length (Man8-12GlcNAc2) with terminal alpha-1,2-linked mannose residues, and not with allergenic terminal alpha-1,3-linked mannose residues as found in other yeasts, especially in the baker’s yeast Saccharomyces cerevisiae.
Biotechnological applications
Ogataea polymorpha with its unusual characteristics provides an excellent platform for the gene technological production of proteins, especially of pharmaceuticals like insulinInsulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
for treatment of diabetes, hepatitis B vaccines or IFNalpha-2a for the treatment of hepatitis C
Hepatitis C
Hepatitis C is an infectious disease primarily affecting the liver, caused by the hepatitis C virus . The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years...
. Derivatives of both CBS4732 and DL-1 are employed in the production of such recombinant compounds. Further yeasts employed for this application are Pichia pastoris, Arxula adeninivorans
Arxula adeninivorans
Arxula adeninivorans is a dimorphic yeast with unusual characteristics. The first description of A. adeninivorans was provided in the mid-eighties. The species was initially designated as Trichosporon adeninovorans...
and Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
and others.
Like other yeasts, O. polymorpha is a microorganism that can be cultured in large fermenters to high cell densities within a short time. It is a safe organ-ism in not containing pyrogens, pathogens or viral inclusions. It can release compounds into a culture medium as it has all the components required for secretion (this is for instance not the case with bacteria like Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
). It can provide attractive genetic components for an efficient production of proteins.
In Fig. 2 the general design of a vector (a genetic vehicle to transform a yeast strain into a genetically engineered protein producer). It must contain several genetic elements:
1. A selection marker, required to select a transformed strain from an untransformed background –this can be done if for instance such an element enables a deficient strain to grow under culturing conditions void of a certain compound like a particular amino acid that cannot be produced by the deficient strain).
2. Certain elements to propagate and to target the foreign DNA to the chromosome of the yeast (ARS and/or rDNA sequence).
3. A segment responsible for the production of the desired protein compound a so-called expression cassette. Such a cassette is made up by a sequence of regulatory elements, a promoter that controls, how much and under which circumstances a following gene sequence is transcribed and as a consequence how much protein is eventually made. This means that the segment following the promoter is variable depending on the desired product – it could be a sequence determining the amino acids for insulin, for hepatitis B vaccine or for interferon. The expression cassette is terminated by a following terminator sequence that provides a proper stop of the transcription. The promoter elements of the O. polymorpha system are derived from genes that are highly expressed, from instance from the MOX gene, the FMD gene or the TPS1 gene mentioned before. They are not only very strong, but can also be regulated by certain addition of carbon sources like sugar, methanol or glycerol.
In 2000 an informal society of scientists was founded named HPWN (Hansenula polymorpha worldwide network) founded by Prof. Marten Veenhuis, Groningen, and Prof, Dr. Gerd Gellissen, Düsseldorf. Every two years meetings are organized.
The attractiveness of the O. polymorpha platform is commercially exploited by several biotech companies for the development of production processes, among others by PharmedArtis, located in Aachen, Germany and the Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK).
External links
- http://www.pharmedartis.de
- http://www.ipk-gatersleben.de/Internet/Forschung/MolekulareZellbiologie/