Why use Pichia Pastoris for recombinant protein production? Advantages and challenges

Choosing an appropriate expression system for recombinant protein production is crucial for the success of a project. The decision largely depends on factors such as the complexity of the target protein, the need for post-translational modifications, and the scalability of the process. Commonly used platforms include bacterial systems like Escherichia coli, yeast systems such as Saccharomyces cerevisiae and Pichia pastoris, as well as mammalian cell systems. In addition, insect cells and transgenic plants are viable alternatives, though they are less commonly used. Each of these systems offers unique advantages and limitations, and selecting the right one requires careful consideration of the specific project requirements.

Yeast systems, including Saccharomyces cerevisiae and Pichia pastoris, are a middle ground between bacterial system and mammalian cells. They combine the scalability and simplicity of microbial systems with the ability to perform some eukaryotic post-translational modifications. Saccharomyces cerevisiae has been widely applied in producing proteins like insulin and vaccines, while Pichia pastoris stands out as a more versatile system. These attributes make yeast systems particularly appealing for biopharmaceutical and industrial applications.

This article will explore the advantages of using Pichia pastoris as an expression system, highlighting its key strengths and addressing the challenges associated with its use. We will also provide comparative analysis with commonly employed production systems like E. coli and mammalian cells to underline their unique position in recombinant protein production.

Advantages of Pichia Pastoris as a recombinant protein production system

Ease of genetic manipulation

A key feature of Pichia pastoris (or Komagataella phaffii) is the facility to manipulate its genome to meet specific production needs¹. The system benefits from well-characterized yeast expression vectors that are simple to design and implement. Pichia pastoris mostly relies on genomic integration for recombinant protein expression. Indeed, this approach ensures stable and consistent gene expression across generations, making it particularly valuable for industrial-scale production². Therefore, these well-characterized vectors enable the stable integration of foreign genes into the yeast genome , often at high copy numbers, to ensure consistent expression across production cycles³.

Additionally, the availability of advanced tools for genetic engineering, such as CRISPR-Cas9, has further expanded the flexibility of Pichia pastoris⁴. This allows researchers to optimize key parameters such as promoter strength, secretion signals, and codon usage, tailoring the system to maximize protein yield and quality¹. The ability to efficiently engineer strains not only accelerates development timelines but also enhances the system's applicability to a wide range of biotechnological challenges.

Scalability for high yields and cost effectiveness

Pichia pastoris is renowned for its exceptional capacity to grow at high cell densities, a feature that makes it particularly suitable for industrial-scale protein production^5,6. When cultivated in optimized bioreactors, Pichia pastoris can achieve protein yields that surpass those of many other microbial or higher eukaryotic expression systems, including bacteria and mammalian cells¹. Its ability to sustain prolonged high-density growth without compromising protein quality is a critical factor in large-scale production. The scalability of Pichia pastoris is further enhanced by its cost-effective growth media, which typically consists of inexpensive substrates such as glycerol or methanol. These cost-efficient inputs reduce overall production expenses, making it a practical choice for commercial applications. Moreover, the yeast's robust growth characteristics ensure consistent performance across different production scales, from laboratory experiments to industrial fermenters⁷. This consistency makes Pichia pastoris a trusted platform for producing recombinant proteins for therapeutic, industrial, and research applications.

Post-Translational Modifications (PTMs)

One of the most significant strengths of Pichia pastoris lies in its ability to perform post-translational modifications (PTMs)⁸, a critical feature not found in bacterial systems. These modifications, such as glycosylation and disulfide bond formation, are essential for producing proteins with correct folding, stability, and therefore biological activity. However, the PTM patterns in Pichia pastoris are not identical to those in other eukaryotes. These differences can impact protein functionality, particularly for therapeutic applications.

- Disulfide Bond Formation

Disulfide bonds are essential for the structural integrity of proteins like hormones, enzymes, and antibodies. The oxidative environment within the endoplasmic reticulum of Pichia pastoris, along with enzymes like protein disulfide isomerase (PDI), ensures proper disulfide bond formation. However, while Pichia pastoris efficiently forms disulfide bonds, its folding efficiency can sometimes differ from mammalian cells. Certain proteins requiring highly specific disulfide arrangements may need further optimization to achieve native-like folding. Despite this, Pichia pastoris remains a powerful system for expressing disulfide-rich proteins⁹.

- Glycosylation

Glycosylation enhances protein folding, solubility, and activity. While Pichia Pastoris can perform N-linked glycosylation, the yeast naturally produces high-mannose structures that differ from the more complex glycosylation patterns found in mammalian cells. Its O-linked glycosylation is similarly limited to short mannose chains rather than the diverse and intricate modifications present in higher eukaryotes¹⁰. To overcome these limitations, genetic engineering has enabled Pichia pastoris to express more human-compatible glycoproteins, broadening its applicability in the pharmaceutical industry¹¹.

Simplified purification

A standout feature of Pichia pastoris is its ability to secrete recombinant proteins directly into the culture medium. This is made possible thanks to a secretion signal integrated into the expression signal. As a result, Pichia pastoris significantly simplifies the process compared to bacterial systems such as Escherichia coli^3,12. Indeed, the yeast does not require cell lysis to retrieve recombinant proteins. By directly secreting them into the culture medium, Pichia pastoris ensures a cleaner starting material for downstream purification, reducing both complexity and cost. Furthermore, the medium contains minimal native proteins¹³. This characteristic reduces both time and costs associated with purification steps.

Broad pH adaptability

The yeast's adaptability is further demonstrated by its ability to grow in a broad acidic pH range of 3.3 to 7.0³. This property is particularly beneficial when pH adjustment is required to minimize the degradation of proteins secreted into the culture medium. By allowing a broad pH variation, it is possible to ensure the stability and integrity of the expressed proteins by minimizing degradation during secretion into the culture medium.

Free from endotoxins

As a yeast-based system, Pichia pastoris offers a critical advantage by naturally avoiding endotoxin contamination. It is a common issue with bacterial systems like Escherichia coli¹⁴. Endotoxins, which are lipopolysaccharides from the outer membranes of Gram-negative bacteria, can provoke severe immune reactions in humans, making their removal essential in pharmaceutical manufacturing¹⁵. Using Pichia pastoris eliminates the need for costly and time-intensive endotoxin removal processes as it streamlines production workflows. This feature not only simplifies regulatory compliance but also enhances the overall safety profile of biopharmaceutical products such as vaccines, therapeutic enzymes, and monoclonal antibodies. Its ability to produce endotoxin-free proteins makes Pichia pastoris a highly reliable platform for clinical and commercial applications.

Addressing challenges in Pichia pastoris

Despite its many advantages, the use of Pichia pastoris is not without limitations. Understanding these challenges can help researchers develop strategies to optimize its use.

Proteolytic activity

Proteolytic activity is one of the primary challenges, as the yeast’s native proteases can degrade recombinant proteins. This issue can be mitigated by using protease-deficient strains³ or optimizing fermentation parameters (e.g. pH) to minimize degradation.

Limited promoter diversity

Another challenge is the limited promoter diversity. The AOX1 promoter is the most commonly used strong methanol-inducible promoter and is highly effective for driving gene expression. However, its limited versatility can present challenges for applications requiring precise control over expression levels. Nevertheless, advancements in promoter engineering have expanded the range of available promoters, providing greater flexibility for customized expression systems¹⁴.

Reliance on methanol

Another potential issue with P. pastoris expression is its reliance on methanol as an inducer when using the AOX1 promoter. While methanol induction allows for strong gene expression, it also introduces several drawbacks. Methanol is highly flammable and requires specialized handling procedures¹⁶. This increases safety risks and operational costs. Additionally, its metabolism demands high oxygen levels, making large-scale fermentation more complex. The metabolic byproducts of methanol utilization, such as formaldehyde and hydrogen peroxide, can also impose stress on the host cells, potentially reducing productivity. To address these challenges, alternative expression strategies have been explored. Researchers have developed methanol-independent promoters which enable protein production without requiring methanol induction. These promoters provide a safer and more cost-effective alternative while maintaining high expression levels. Additionally, strain engineering efforts have led to the development of methanol-negative P. pastoris variants that can still achieve robust protein yields¹⁷. These strains are designed to optimize carbon metabolism, allowing for efficient protein expression using alternative carbon sources.

Overall, why choosing Pichia Pastoris among the most commonly used expression system?

The choice of an expression system is one of the most critical decisions in recombinant protein production, as it directly impacts the quality, yield, cost, and scalability of the process. We will display here a comparison between the three most common systems: Pichia pastoris, Escherichia coli, and mammalian cells. They each offer distinct strengths and limitations, making them suitable for different types of proteins and production goals.

For simpler proteins without post-translational modifications, bacterial systems like E. coli are often preferred due to their rapid growth, straightforward genetic manipulation and cost-effectiveness¹. However, they are unable to perform eukaryotic post-translational modifications, which limit their use for more complex therapeutic proteins. On the other hand, mammalian cells are best suited for producing complex proteins that require human-like modifications, though they come with higher costs and longer production timelines. Pichia pastoris bridges the gap. This yeast offers a eukaryotic environment for post-translational modifications and proper protein folding, combined with the scalability and cost advantages of microbial systems.

The versatility of Pichia pastoris (now Komagataella phaffii) makes it suitable for a wide range of applications:

1. Therapeutic Proteins: Antibodies, hormones, and vaccines benefit from its ability to perform post-translational modifications.

2. Industrial Enzymes:Used in food processing, detergents, and biofuels, Pichia pastoris provides high yields at low cost.

3. Research Tools: Structural proteins for crystallography and functional studies are easily produced with this system.

Conclusion

Pichia pastoris stands out as an optimal expression system, combining the scalability and efficiency of microbial platforms with the ability to perform essential post-translational modifications. Its high protein yields, cost-effective production, and secretion capabilities make it a valuable choice for biopharmaceutical and industrial applications.

At Eurogentec, we have developed a high-yield and low O-glycosylation platform for Pichia pastoris-based protein production. Our optimized expression system minimizes unwanted glycosylation while ensuring high-quality recombinant protein yields.

Discover our poster on high-yield and low O-glycosylation Pichia productionto see how we optimize protein expression for maximum efficiency.

Our expertise spans strain development, process optimization, and large-scale manufacturing, providing a reliable and flexible solution for therapeutic, diagnostic, and research applications.

Discover how our Pichia pastoris-based production services can support your project, or explore our GMP recombinant protein manufacturing capabilities for high-quality, scalable production.

1. Tripathi, N. K. & Shrivastava, A. Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development. Front. Bioeng. Biotechnol. 7, 420 (2019).
2. Vogl, T., Gebbie, L., Palfreyman, R. W. & Speight, R. Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris. Appl. Environ. Microbiol. 84, e02712-17 (2018).
3. Li, P. et al. Expression of Recombinant Proteins in Pichia Pastoris. Appl. Biochem. Biotechnol. 142, 105–124 (2007).
4. Weninger, A., Hatzl, A.-M., Schmid, C., Vogl, T. & Glieder, A. Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J. Biotechnol. 235, 139–149 (2016).
5. Julien, C. Production of Humanlike Recombinant Proteins in Pichia pastoris. (2006).
6. Shemesh, P. & Fishman, A. Optimal fermentation conditions for growth and recombinant protein production in Pichia pastoris: Strain selection, ploidy level and carbon source. Curr. Res. Food Sci. 9, 100840 (2024).
7. Liu, W.-C. et al. Scaling-up Fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci. Rep. 6, 18439 (2016).
8. Karbalaei, M., Rezaee, S. A. & Farsiani, H. Pichia pastoris : A highly successful expression system for optimal synthesis of heterologous proteins. J. Cell. Physiol. 235, 5867–5881 (2020).
9. Ma, Y., Lee, C.-J. & Park, J.-S. Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds. Antibiotics 9, 541 (2020).
10. Irani, Z. A., Kerkhoven, E. J., Shojaosadati, S. A. & Nielsen, J. Genome‐scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins. Biotechnol. Bioeng. 113, 961–969 (2016).
11. Liu, C.-P. et al. Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation. Proc. Natl. Acad. Sci. 115, 720–725 (2018).
12. Tachioka, M. et al. Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris. Biotechnol. Biofuels 9, 199 (2016).
13. Balamurugan, V., Reddy, G. R. & Suryanarayana, V. V. S. Pichia pastoris: A notable heterologous expression system for the production of foreign proteins—Vaccines. INDIAN J BIOTECHNOL (2007).
14. Pan, Y., Yang, J., Wu, J., Yang, L. & Fang, H. Current advances of Pichia pastoris as cell factories for production of recombinant proteins. Front. Microbiol. 13, 1059777 (2022).
15. Mamat, U. et al. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microb. Cell Factories 14, 57 (2015).
16. Shen, W. et al. A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb. Cell Factories 15, 178 (2016).
17. Wang, J. et al. Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris. Sci. Rep. 7, 41850 (2017).