Ogataea angusta

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Ogataea angusta
Systematics
Subdivision : Saccharomycotina
Class : Saccharomycetes
Order : Real yeast (Saccharomycetales)
Family : Saccharomycetaceae
Genre : Ogataea
Type : Ogataea angusta
Scientific name
Ogataea angusta
( Teun. , HH Hall & Wick. ) Suh & Zhou

Ogataea angusta (mostly still known by the synonyms Pichia angusta or Hansenula polymorpha ) is a methylotrophic yeast species with unusual properties. It is used as a protein factory for pharmaceutical products and is one of a limited number of methylotrophic yeasts (yeasts thatcan growon methanol , formerly also called methyl alcohol).

Basics

Other yeast species with this ability are Candida boidinii , Pichia methanolica, and Komagataella phaffii . Taxonomically, P. angusta belongs to the Saccharomycetaceae family . In more recent taxonomy books the genera Pichia and Hansenula were merged, and H. polymorpha was renamed Pichia angusta . However, many scientists want the popular name H. polymorpha to be retained. But after it was established that Pichia is polyphyletic , the species was placed in the genus Ogataea and is therefore validly called Ogataea angusta . There are three different tribes of this type of unexplained relationship and independent origin. They have been discovered in soil samples, in the intestines of insects and in rotten orange juice since the beginning of the 1950s . They have different properties and are either popular objects in basic scientific research or are used after genetic modification for the production of proteins:

  • Strain CBS4732 (CCY38-22-2; ATCC34438, NRRL-Y-5445)
  • Strain DL-1 (NRRL-Y-7560; ATCC26012)
  • Strain NCYC495 (CBS1976; ATAA14754, NRLL-Y-1798).

The strains CBS4732 and NCYY495 can be crossed with each other, strain DL-1 is not crossable with the other two. The strains CBS4732 and DL-1 are used for the genetic engineering production of proteins, strain NCYC495 is mainly used for studies of nitrate assimilation. The genome of CBS4732 has been completely sequenced.

Fig. 1: (A) Electron microscopic image of a budding “H. polymorpha ”cell that was cultivated in the chemostat with methanol as the carbon source. It contains large peroxisomes that fill the inside of the cell. (modified from Gellissen et al. 2005; photo by Prof. Veenhuis, Groningen).

"H. polymorpha “ is a thermo-tolerant microorganism - some strains can still grow at temperatures above 50 ° C. In addition, the yeast can assimilate nitrate (this is very unusual for a yeast) and grow on different sugars , glycerine or even methanol (the better known baker's yeast only grows on grape sugar and turns it into alcohol ). Cells that grow at elevated temperatures accumulate trehalose , a sugar found in insects, and use it to protect against heat. Trehalose is not necessary for growth, but it is necessary for the acquisition of thermal tolerance. The steps necessary for the synthesis of trehalose were elucidated for this type of yeast and TPS1 , the gene for the key enzyme in the synthesis pathway, was isolated and characterized. All methylotrophic yeast species have an identical metabolic pathway for the use of methanol. The growth on methanol is accompanied by a massive increase in peroxisomes , certain cell organelles (Fig. 1).

The first steps in methanol metabolism take place in them. "H. polymorpha “ is a model organism for studying the functions of peroxisomes and the molecular biology on which they are based. During growth on methanol, certain metabolic pathway enzymes are produced in large quantities, including MOX (methanol oxidase), FMDH (formate dehydrogenase) and DHAS (dihydroxyacetone synthase). Their presence is regulated by controlling the transcription (formation of mRNA) of the corresponding genes. In the related yeast species C. boidinii , P. methanolica , and K. phaffii ( Pichia pastoris ) mentioned above , this gene expression is strictly dependent on the presence of methanol, while in “H. polymorpha “ this is also caused by suitable quantities of glycerine or under sugar starvation conditions (glucose starvation).

Fig. 2: (B) Methanol metabolism in “H. polymorpha ” (modified from Gellissen et al. 2005). 1 - alcohol oxidase, 2 - catalase, 3 - dihydroxyacetone synthase, 4 - formaldehyde dehydrogenase, 5 - formate dehydrogenase, 6 - dihydroxyacetone kinase, 7 - GSH – glutathione, Xu5P - xylulose-5-phosphate, FBP - fructose-1,6 bisphosphate.

Pichia angusta produces glycoproteins (proteins to which sugar molecules are attached in a chain) - there are N- and O-linked sugar chains. The terminal sugars (mannose) are linked in N-chains with alpha-1,2 bonds and not with potentially allergenic alpha-1,3 bonds such as. B. in the baker's yeast Saccharomyces cerevisiae .

Pichia angusta offers an excellent platform for the genetic engineering production of proteins, especially pharmaceuticals such as insulin for diabetics, for hepatitis B vaccines or for IFN alpha-2a for the treatment of hepatitis C.

The previously described unusual properties make “H. polymorpha ”has become an attractive platform for the genetic production of proteins. Based on genetically modified "H. polymorpha “ yeasts there are already numerous products and production processes (some important examples are given in the title). Descendants of the strains CBS4732 and DL-1 are used for this. Other types of yeast for this application include K. phaffii ( Pichia pastoris ), Arxula adeninivorans , Saccharomyces cerevisiae and others.

Yeasts are microorganisms that can be cultivated to high cell densities in large fermenters in a short time. "H. polymorpha “ is a safe organism because it does not contain any pyrogenic or pathogenic substances. Yeasts can release (secrete) proteins into the medium, since they have the necessary structures of higher cells, such as those of humans. The intestinal bacterium cannot do this, for example. "H. polymorpha “ provides attractive genetic elements for the efficient production of proteins.

Fig. 3 shows the general scheme of a vector (a circular DNA molecule for transforming a yeast strain into a genetically modified protein producer). Such a vector (also known as a plasmid) must contain various genetic elements:

  1. A "selection marker" that is necessary to recognize and select a transformed strain from a non-transformed background - this can e.g. This can be achieved, for example, when such a genetic element enables an originally deficient strain to grow under culture conditions in which a substance essential for survival, such as a certain amino acid, is missing, but which this strain itself can no longer produce.
  2. Certain genetic elements for the propagation of the DNA and for the targeted smuggling of the foreign DNA into a certain position on the chromosomes of the receiving yeast cell (ARS and / or rDNA sequence).
  3. A segment that is necessary for the production of the desired protein - an expression cassette . Such a cassette consists of a sequence of regulatory elements: first of all, it contains a promoter that controls how much and under what circumstances a subsequent sequence is transcribed (an mRNA copy is produced), and ultimately how much and under what circumstances a desired protein will be produced. The following segment is variable depending on the protein to be produced - it can be a gene sequence with the definition of the amino acid sequence for insulin, hepatitis B antigens or interferon. The expression cassette is closed by a terminator, which brings about a correct conclusion of the transcription process. The promoter elements of the H. polymorpha system are derived from genes that are strongly expressed, e.g. B. of the already mentioned MOX , FMD or TPS1 genes. The properties of these promoters such as strength and controllability through certain carbon sources are retained.
Fig. 3: Basic structure of a vector. This basic vector contains all elements for the replication of the plasmid in the E. coli system and a multicloning site (MCS) for the integration of modules for ARS, rDNA, selection markers and expression cassettes. For this purpose, the ARS fragments were flanked with the restriction sites for Sac II and Bcu I, the rDNA region with Bcu I and Eco 47III, the selection markers with Eco 47III and Sal I and the promoter elements with Sal I and Apa I.

application

The Pichia angusta platform is used by various biotechnology companies to develop production processes for important proteins, including ARTES Biotechnology GmbH in Langenfeld, PharmedArtis in Aachen and the Leibniz Institute for Plant Genetics and Crop Plant Research (IPK).

HPWN ( Hansenula polymorpha worldwide network)

Many academic research groups around the world are studying this organism. In 2000, a scientific society was founded under the name HPWN ( Hansenula polymorpha worldwide network) by Prof. Dr. Marten Veenhuis, Groningen, and Prof, Dr. Gerd Gellissen, Düsseldorf, founded. Scientific meetings are organized every two years.

Individual evidence

  1. ↑ Cletus Kurtzman, JW Fell, Teun Boekhout (Ed.): "The Yeasts: A Taxonomic Study, Volume 1"; Elsevier, BV 2010: p. 685 ff .; ISBN 978-0-444-52149-1 .
  2. ↑ Gerhard Steinborn, Erik Böer, Anja Scholz, Kristina Tag, Gotthard Kunze, Gerd Gellissen: Application of a wide-range yeast vector (CoMed ™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts . In: Microbial Cell Factories . tape 5 , 2006, ISSN  1475-2859 , pp. 33 , doi : 10.1186 / 1475-2859-5-33 .
  3. ↑ Yeast Genetics Working Group. IPK , archived from the original on February 24, 2015 ; Retrieved May 22, 2013 .
  4. ↑ In autumn 2008 this meeting is in Estonia. Information on this society and the conferences will be available from summer 2007 at www.hansenula-network.com.

literature

  • Gellissen G (Ed) (2002) Hansenula polymorpha - biology and applications. Wiley-VCH, Weinheim ISBN 978-3-527-60235-3
  • Gellissen G (Ed) (2005) Production of recombinant proteins - novel microbial and eukaryotic expression systems. Wiley-VCH, Weinheim ISBN 978-3-527-31036-4