Pichia pastoris Fermentation Services

BOC Sciences can provide a variety of microbial fermentation services using Pichia pastoris as host cells, including strain improvement, fermentation process optimization, microbial fermentation production. With our complete strain development platform and extensive experience in microbial fermentation, we are able to offer one-stop fermentation services ranging from laboratory fermentation to microbial manufacturing. Due to the potency of Pichia pastoris as an expression system for protein production, we utilize Pichia pastoris for microbial fermentation and applications in vaccines, recombinant proteins, antibody fragments, cytokines, and other fields. Our Pichia pastoris system is an efficient industrial tool that provides our customers with cost-effective microbial manufacturing.

What is Pichia pastoris?

Pichia pastoris, abbreviated as P. pastoris, is a methanotrophic yeast, a heterotroph discovered in the 1960s. P. pastoris can survive and reproduce using different carbon sources, such as methanol, glucose, glycerol. P. pastoris exists in two cell types, haploid and diploid cells, obtained through mitosis, sporulation and meiosis, respectively. P. pastoris is a single-celled eukaryote. That being said, proteins within P. pastoris can be studied and compared homologously with other more complex eukaryotic species to understand their function and origin.

The P. pastoris has become an advanced heterologous gene expression system and is widely used to produce various heterologous proteins. The production of P. pastoris recombinant proteins is controlled by the methanol-inducible alcohol oxidase 1 (AOX1) promoter, which is characterized by methanol as a carbon and energy source. Due to its ability to serve as a model organism for genetic studies and as an protein expression system, P. pastoris has become an essential microbial strain for biological research and applications. For example, P. pastoris can functionally process large molecular weight proteins, which is useful in translation hosts.

Function of Pichia pastoris in fermentation

P. pastoris has potential characteristics that are leading to the development of several components that might enhance the overall bioprocess's efficiency. The most widely used technique for producing heterologous proteins uses the AOX1 promoter, which is inhibited in cells cultured on glycerol. In the event that methanol is the only carbon source in the media, it is induced around 1000 times. But, as was the case with the methanol-free GAP promoter, it is crucial to choose a promoter that does not inhibit P. pastoris growth when it is grown in glycerol. It has been noted that P. pastoris produces recombinant proteins that are often nicely folded and that it is devoid of viral nucleic acids, potentially oncogenes (which are occasionally discovered in mammalian cells), and harmful cell wall pyrogens (found in Escherichia coli). Glycerol is frequently utilized as the primary starting carbon source for P. pastoris fermentations. Candida antarctica (CalB) produces the 33 kDa globular protein lipase B, which has an active site composed of Ser–Asp–His and Sn-3 regiospecificity for triacylglycerol. Due to these characteristics, it is now one of the lipases that is most often utilized in biocatalytic processes. But for this purpose, the Pichia pastoris (Komagataella phaffii) cell factory has shown to be a great substitute during the past ten years. Because of its capacity to thrive at high cell densities on straightforward, affordable medium and its openness to post-translational modification using a range of potent promoters, this methylotrophic yeast is mostly utilized as a host for recombinant proteins.

Heterologous protein production in Pichia pastoris. (de Macedo Robert J., et al., 2019)

Effects of the different fermentation parameters on P. pastoris

A recent research examined the effects of crude glycerol impurities on P. pastoris yeast lipase synthesis. These impurities included methyl ester, grease, glycerol, methanol, and metal ions Na⁺, Ca²⁺, and Fe³⁺. Under the same fermentation circumstances, the use of crude glycerol instead of pure glycerol boosted cell growth and enzyme activity, and impurities accelerated the stationary phase. According to these findings, pollutants may hasten P. pastoris proliferation.

P. pastoris is capable of utilizing other hexose and pentose sugars, such as fructose, rhamnose, mannose, trehalose and also various sugar alcohols, including D-mannitol and sorbitol. Utilization of ethanol, lactate, succinate, lactic acid, acetic acid, formate, succinic acid, citric acid, gluconate, alanine, oleic acid, and acetate are also proved. On the contrary, P. pastoris is unable to consume galactose, L-sorbose, lactose, sucrose, maltose, cellobiose, melibiose, raffinose, melezitose, inulin, soluble starch, L-arabinose, D-arabinose, D-ribose, D-glucosamine, N-acetyl-D-glucosamine, ribitol, methyl α-D-glucoside, salicin, D-gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate, saccharate.

P. pastoris is reported to grow in a wide variety of pH values. Because host proteases are present during Pichia fermentation, the low response might be the result of proteolytic destruction in this pH range. Since the pH range in which P. pastoris grows is 3 to 7, it is conceivable that protein synthesis would decline as pH rose.

The majority of Pichia yeast fermentation procedures are conducted around 30 °C, which is the ideal temperature. Temperatures beyond 32 °C have been shown to reduce the production of some proteins and cause cell death in cultures by causing cell lysis and elevated levels of protease activity.

Glycerol metabolism in P. pastoris is aerobic, with 0.5-1.6 vvm flux even at high glycerol concentrations in fed-batch settings, despite the fact that glycerol is a poor fermentative carbon source. The experimental circumstances indicate that an aeration rate increase of up to 1 vvm is sufficient for fed-batch culture of P. pastoris.

Different carbon sources for P. pastoris cultivation. (Ergün B G., et al., 2022)

Scheme for the interdependency of P. pastoris HCDF automation process. (Liu W C., et al., 2019)

Fermentation process of Pichia pastoris

Thawing a functioning cell bank vial into a 2 L Erlenmeyer flask with the inoculum expansion media is the first step in the fermentation process. The flask is shaken and incubated until the required optical density (OD) is obtained during the inoculum formation phase. After the inoculum flask is added to the production fermentor, it is cultured in batch phase until the optical density (OD) reaches a predetermined target. Next, in order to restrict the concentration of glucose in the broth and encourage oxidative development, Feed 1 is introduced at a predetermined pace. The culture pH and temperature are maintained at the same predetermined levels during the batch and fed-batch development phases. Again, the induction phase begins upon the achievement of a predefined OD goal. The process's two different phases, the adaptation phase and the production phase, make up the induction phase. Feed 2 with the inducer is introduced during the adaptation period, in addition to Feed 1. Feed 1 is stopped when the adaptation phase's duration hits a predefined threshold, and Feed 2's flow rate is changed to encourage the production of new product. To lessen the possibility of proteolysis, the culture temperature is lowered at the start of the induction phase.

Process flow diagram of the P. pastoris fermentation steps from vial thaw to harvest by centrifugation. (Harms J., et al., 2008)

Advantages of P. pastoris Fermentation

• Culture: P. pastoris can be grown on simple, inexpensive media with fast growth rates. P. pastoris is a methylotrophic bacterium, suggesting that it can be grown with simple methanol as the sole energy source. P. pastoris can also be grown in shake flasks or fermenters, making it suitable for small-scale and large-scale production.
• Growth: P. pastoris, like other widely used yeast models, has a relatively short lifespan and fast regeneration time. P. pastoris can be grown in medium with high cell densities. This feature is compatible with the expression of heterologous proteins and allows the production of higher yields.
• Production: The P. pastoris expression system is capable of producing high concentrations of heterologous proteins. P. pastoris is easy to manipulate genetically, and a wide range of vectors and strains are available. This eukaryote also has the potential to produce soluble and correctly folded recombinant proteins with post-translational modification (PTM), such as N-glycosylation. The target protein function may require eukaryotic processing that is not available in prokaryotes.

Applications of P. pastoris Fermentation

• P. pastoris is a model organism used for genomic studies and genetic analysis. It is a single-cell eukaryote capable of complex eukaryotic genetic processing in a relatively small genome. Due to its ability to recombine with foreign DNA and process large proteins, P. pastoris can be engineered as a transformation host for new proteins production.
• Based on the characteristics of P. pastoris, it has been successfully used to produce a large number of biopharmaceuticals and industrial enzymes. In the pharmaceutical industry, high cell density fermentation of P. pastoris enables the production of large amounts of highly active and low-cost recombinant proteins. In addition, P. pastoris is capable of proteins glycosylation for biotherapeutics. In the food industry, P. pastoris is used to produce different kinds of enzymes as food additives with many functions.

Project Workflow

• Customer advisory
• Project discussion
• P. pastoris served as host cell
• Strain improvement and fermentation development
• Novel strain evaluation
• Project delivery

References

1. Enikö Zörgö, et al. Ancient evolutionary trade-offs between yeast ploidy states, PLoS Genet, 2013, 9(3):e1003388.
2. Wan-Cang Liu, et al. Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production, Crit. Rev. Biotechnol, 2019, 39, 258-271.
3. Corinna Rebnegger, et al. Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates, Appl Environ Microbiol, 2016, 82(15):4570-4583.
4. Palmerín-Carreño D., et al., Optimization of a recombinant lectin production in pichia pastoris using crude glycerol in a fed-batch system, Processes, 2021, 9(5): 876.
5. de Macedo Robert J., et al., Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris, Biochemical engineering journal, 2019, 147: 39-47.
6. Liu W C., et al., Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production, Critical reviews in biotechnology, 2019, 39(2): 258-271.
7. Harms J., et al., Defining process design space for biotech products: case study of Pichia pastoris fermentation, Biotechnology progress, 2008, 24(3): 655-662.
8. Ergün B G., et al., Second generation Pichia pastoris strain and bioprocess designs, Biotechnology for Biofuels and Bioproducts, 2022, 15(1): 150.