Pichia pastoris

Komagataella pastoris ( usually still under the synonym: Pichia pastoris is known ) is a methylotrophic yeast species, which is one of the most expression systems of biotechnology. At the same time P. pastoris is an important model organism for research.

An advantage over other organisms represents mainly the ability to utilize methanol as a sole energy and carbon source ( methylotroph ). Methanol is cheaper than conventional nutrient media which are based on various saccharides. Furthermore, P. pastoris can be grown in very high cell densities.

P. pastoris in biotechnology

P. pastoris was first introduced in the 1970s by the Phillips Petroleum Company (now ConocoPhillips ) cultivated on a commercial scale and sold directly as a protein-containing animal feed. Interest in the yeast was aroused mainly by its methylotrophic character, since methanol was very cheap compared to other nutrient media. Due to the oil crisis in 1973, the price of methanol increased significantly, while at the same time, the price of soybeans, the largest alternative source for animal feed dropped. The use of P. pastoris was thus for a long time no longer of economic importance. Only through progressive research in the field of genetics and the development of recombinant DNA technology (genetic engineering ) was over time P. pastoris as an expression system increasingly popular. Since the yeast of the U.S. Administration, the Food and Drug Administration ( FDA), the so-called GRAS status (general regarded as safe; generally regarded as safe ) received, it has also been established for the production of active pharmaceutical ingredients. This is, inter alia, the fact due to that disease-causing factors such as pyrogens and lysogenic viruses in yeast non-existent.

Currently, more than 500 pharmaceutical products and other recombinant proteins are cloned and produced in P. pastoris. The following table shows some examples of the expression organism of foreign proteins in P. pastoris are:

The Pichia pastoris expression system

The use of methanol due to the presence of proteins, which can inject alcohol into the metabolism of the yeast. In P. pastoris, these are mainly the alcohol oxidases I and II ( AOXI, AOXII ), which oxidize as initial enzymes of methanol to hydrogen peroxide and formaldehyde. But especially the alcohol oxidase I has a very low affinity for oxygen; the yeast compensate this drawback by adding large amounts of AOXI it forms (up to 30 % of cell protein). For this to be possible only subject to the AOXI gene control of the very strong AOX promoter, leading only at presence of methanol in the transcription of the corresponding gene. This results in a promoter which can be induced by the addition of methanol. If one assumes now the gene sequence of a recombinant protein of the control of a promoter AOX (usually AOX1 ), allows the accumulation of biomass is separated from the actual protein production in a fermentation process, which is usually sought. Only by the addition of methanol as inductor the AOX promoter is read and thus synthesized the recombinant protein. In addition, P. pastoris can be cultured over a wide pH range of time, without the growth is significantly impaired. Post-translational modifications such as formation of disulfide bridges or glycosylation of proteins ( the attachment of specific sugar residues ) can be carried out by the yeast. Pichia can also secrete proteins very efficiently, ie them out of the cell, subsequent processing steps ( downstream processing ) makes it easier. In P. pastoris for use next vectors, which are those genetic constructs carrying the recombinant protein are usually integrated into the host genome.

Glycosylation and " humanization "

Especially the Glykosylierungsmechanismus of yeasts is a recurring problem in the expression of human, pharmaceutical agents dar. Usually produced in yeast proteins hypermannosyliert, i.e. up to 200 mannose residues attached thereto ( in P. pastoris: 8-14 mannose residues). These structures differ in each case, substantially from the glycosylation from human, which is of a complex type. So-called " hypermannosylierte " proteins are partially inoperative in the human body, can cause allergic reactions and have a very short half -life in human serum, since they va. are bound by mannose receptors in the liver. In order to produce fully glycosylated human proteins in a high -density fermentation of P. pastoris, in recent years at the " humanization " ( humanization ) of glycosylation was researched. Basically, unwanted enzymes of yeast metabolism, for example, Hypermannosylation responsible for drawing, removed while genes that are involved in human glycosylation can be introduced by recombinant DNA techniques into the organism. 2004 could be produced in its natural, human glycosylation in P. pastoris strain as the first time the human protein erythropoietin ( EPO). The aim of further research in this area is the application of the results to other expression systems as well as the increase in the expression rates.

Pichia pastoris

P. pastoris in biotechnology

The Pichia pastoris expression system

Glycosylation and " humanization "

Footnotes

Further Reading