A Brief Guide to Pichia pastoris Fermentation

What is pichia pastoris and their functions?

Pichia pastoris is a type of yeast that is commonly used in biotechnology for the production of recombinant proteins. It is known for its ability to efficiently produce heterologous proteins with high yields. Pichia pastoris offers several advantages for protein production:

Strong and tightly regulated promoters: Pichia pastoris has strong promoters, such as the alcohol oxidase (AOX) promoter, which can drive high levels of protein expression. The AOX promoter can be induced by methanol and tightly regulated, allowing for precise control of protein production.

High protein secretion: Pichia yeast has a natural ability to secrete proteins into the extracellular medium, simplifying downstream purification processes.

Growth on simple media: Pichia pastoris can grow on inexpensive, chemically defined media, reducing production costs.

Post-translational modifications: Pichia pastoris is capable of carrying out proper folding and post-translational modifications, such as glycosylation, disulfide bond formation, and proteolytic processing, leading to the production of biologically active and stable proteins.

Scalability: The fermentation process of Pichia yeast can be easily scaled up from laboratory-scale to industrial-scale production.

Due to these advantages, Pichia pastoris has become a popular choice for the production of a wide range of proteins, including enzymes, antibodies, vaccines, and industrial proteins.

The Pichia pastoris fermentation process in 6 steps

1. Inoculum preparation: A small amount of Pichia pastoris cells from a stock culture is inoculated into a small volume of growth medium and incubated at an optimal temperature and pH for growth. This step allows the cells to multiply and reach a sufficient density for inoculation into the main fermentation culture.

2. Seed culture expansion: The inoculum from the previous step is used to inoculate a larger volume of growth medium in a shake flask or a bioreactor. The seed culture is incubated under controlled conditions (such as agitation and aeration) to further increase the cell density.

3. Main fermentation: The seed culture is used to inoculate a larger volume of production medium in a bioreactor. The production medium usually contains a carbon source (such as methanol) that triggers the expression of the target protein in Pichia pastoris. The fermentation parameters, such as temperature, pH, dissolved oxygen, and agitation rate, are carefully controlled to optimize cell growth and protein expression.

4. Induction phase: Once the cell density reaches a certain threshold, the carbon source in the production medium is switched to methanol, which acts as an inducer for protein expression. Methanol is typically added gradually to prevent cell stress and maintain optimal conditions for protein production.

5. Harvesting: After a certain period of induction, the fermentation is stopped, and the cells are harvested. This usually involves separating the cells from the fermentation broth, either by centrifugation or filtration. The harvested cells can be further processed to extract and purify the target protein.

6. Downstream processing: The harvested cells are typically disrupted to release the target protein, followed by various purification steps to isolate and purify the protein of interest. This may include steps such as cell lysis, precipitation, chromatography, and filtration.

The fermentation process for Pichia pastoris can be optimized and scaled up for the production of large quantities of recombinant proteins. It is a versatile expression system widely used in biotechnology and industrial applications.