Team:Georgia State/WhyPichia




Pichia Vs Saccharomyces

Greater Cell Concentrations The preference of Pichia pastoris for respiratory growth is a key characteristic which allows it to be cultured at high cell densities (500 OD600 U ml31)[1]. This trait lends an advantage to Pichia over Saccharomyces cervisiae: Resultant ethanol from S. cervisiae fermentation accumulates which hinders culture growth and therefore protein production. High levels of cell growth in fermenter cultures are vital to foreign protein production since concentration of the secreted product in "extracellular medium is...proportional to the concentration of cells in the culture". [2]

General notworthy differences inlcude the relatively stable ell membrane lipids of Pichia that form etter biocatalyst.(jiang, 2008). Pichia also has more compact and organized golgi bodies than Saccharomyces(Morawski, 2000). In contrast, there are many similarities between Pichia and Saccharomyces that greatly increase the benefits affiliated with using Pichia. Genetic similarity between the two organisms has enabled expression of similar genes and compatibility between vectors. Other similarities include identical pathways for TCA, pentose phosphate, glycolysis and amino acid synthesis(Fiaux, 2003) and (Baumann, 2010).

Easy and Inexpensive to Culture

The components of P. pastoris media (glycerol, methanol, salts, trace elements, and biotin) are relatively inexpensive and as such are well suited for large-scale heterologous protein production. The common protocol is to allow P. pastoris to accumulate biomass in a glycerol/carbon-source medium while foreign protein expression is repressed. Once glycerol stocks have been depleted, methanol is added to induce expression. Any undesirable protease activity can be easily averted by adjusting pH. Pichia's broad spectrum of optimal growth pH (3.0-7.0) provides ample wiggle room. A number of other techniques may be employed in minimizing proteolysis including lowering growth rate via methanol-level modulation, addition of protease inhibitors, addition of alternative protease substrates, and even simply lowering process temperature.

High production of foreign Protein while low levels of endogenous protein

Extracellular expression of foreign proteins is favorable to intracellular production due to Pichia's extremely low levels of endogenous protein secretion, which makes it easier to isolate the protein of interest. Pichia vectors can also be cloned with foreign genes which align with a secretion signal (native to the protein, native to Pichia, or native to Saccharomyces), facilitating extracellular production. However, intracellular protein production is also high due to the efficiency--at carefully monitored methanol levels--of the AOX promoter.

a. Large Upscale of Proteins

Pichia pastoris is greatly used as a protein expression system because of several distinctive reasons. This methylotrophic yeast, P. pastoris, has a high growth rate and is able to grow on uncomplicated, low-cost medium. The presence of two strongly inducible promoters (AOX I & AOXII) allows P. pastoris to use methanol as its sole carbon and energy source. Large upscale (<130 g/l dry cell weight) can be maximized because of its preference for respiratory growth, which allows it to be cultured at high cell densities compared to the fermentative yeasts such as Saccharomyces cerevisiae. (Weidner et al). In turn, this allows for large yields of the recombinant proteins to be produced. The protein production of P. pastoris is largely extracellular because the amounts of endogenous proteins are limited. Secretion in this manner is in fact the first step of purification since the medium used to grow P. pastoris has no added proteins.

Pichia pastoris versus Esherichia coli

The bacterium E. coli has been frequently chosen as a host to express recombinant, heterologous proteins. Although many successful experiments have used E. coli as their expression system, P. pastoris has become an increasingly popular alternative due to the shortcomings of E. coli. P. pastoris serves as a better host when attempting to express foreign genes because this yeast can perform post-translational modifications and fold proteins properly, tightly regulate transcription via its wide range of promotors, and be purified easily.

a. Post-translational modifications

Unlike P. pastoris, E. coli cannot perform higher eukaryotic post-translational modifications. As a result, proteins requiring these modifications often fold incorrectly when produced by E. coli. These disadvantages, which result from E. coli’s prokaryotic nature, limit the types of proteins that this system can express. Proteins that contain disulfide bonds or require post-translational modifications such as glycosylation, isomerization, or phosphorylation are not always properly expressed; they can be insoluble or improperly folded, which require additional solubilization and re-folding steps (Daly et al). Take for instance erythropoietin (EPO), a glycosylated protein. When EPO is expressed in E. coli, it is not glycosylated and becomes less resistant to unfolding compared to its natural, glycosylated form (Daly et al). Therefore, additional steps are needed in order to form a stable protein, which is time-consuming, costly, and has the tendency to produce low yields.

A study done by Leuking et al. also supports the use of P. pastoris for protein expression. Vectors were created for both E. coli and P. pastoris using multiple cDNAs from a human fetal brain expression library. Out of the 29 DNA clones, all produced soluble proteins in P. pastoris, while E. coli was much less successful. In E. coli, only nine produced soluble proteins; 15 were detected as inclusion bodies; and five were not expressed at all. These differences are likely due to E. coli’s lack of eukaryotic abilities to perform post-translational modifications and properly fold proteins (Leuking et al).

b. Powerful promoter systems

P. pastoris’s wide range of promotors also aid in its use for foreign protein production. It has many strongly induced promotors, which allow sufficient expression of the genes of interest and therefore, a high concentration of proteins can be easily and inexpensively produced (Weidner et al). The alcohol oxidase genes, AOX1 and AOX2, are frequently used promotors because they are easily induced by methanol; because P. pastoris is methylotropic, meaning it can metabolize methanol as its carbon and energy source, adding methanol to the growth medium will induce these two promotors and then express the desired protein (Macauley-Patrick et al). High concentrations of protein can then be produced. A third advantage that P. pastoris has over E. coli is that proteins expressed by P. pastoris are easily purified (Weidner et al). Because P. pastoris secretes its recombinant protein in the growth medium and secretes low levels of endogeneous proteins, there is a better chance of obtaining high yields of uncontaminated proteins in comparison to E. coli (Weidner et al). In essence, there are fewer steps required to obtain the desired protein.

Works Cited Daly, Rachel, and Milton T. W. Hearn. "Expression of Heterologous Proteins in Pichia pastoris: a Useful Experimental Tool in Protein Engineering and Production." PubMed, 26 Nov. 2004. Web. 25 Oct. 2010. <>. Leuking, Angelika, Caterina Holz, Christine Gotthold, Hans Lehrach, and Dolores Cahill. “A System for Dual Protein Expression in Pichia pastoris and Escherichia coli.” PubMed, 15 Dec. 2003. Web. 25 Oct. 2010. <>. Macauley-Patrick, Sue, Mariana L. Fazenda, Brian McNeil, and Linda M. "Heterologous Protein Production Using the Pichia pastoris Expression System." PubMed, 22 Mar. 2005. Web. 25 Oct. 2010. <>. Weidner, Maria, Marcus Taupp, and Steven J. Hallam. "Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris." PubMed, 25 Feb. 2010. Web. 25 Oct. 2010. <>.