Pichia is a genus of yeast that has recently gained attention in the field of synthetic biotechnology due to its high protein secretion and post-translational modification capacity. It’s a promising host for producing various proteins, enzymes, biofuels, and pharmaceuticals. Researchers have developed an array of genetic tools and strategies to optimize Pichia strains for specific applications, such as improving protein yields, glycosylation patterns, and resistance to environmental stress. Additionally, Pichia can be engineered for the production of non-protein products, such as carotenoids and terpenoids. Overall, the synthetic biotechnology application of Pichia demonstrates the potential of this yeast genus as a versatile platform for biomanufacturing now, and in the future.
A Brief Background of the Genus Pichia
Figure 1: Pichia fermentans cell: (A) image using phase contrast microscopic (B) image from scanning electron microscope, (C) cells attached on a solid surface, (D) yeast biofilm
Gene editing with the genus Pichia is a relatively recent development in the field of synthetic biotechnology. The use of Pichia as a host for recombinant protein production was first reported in 1985. Pichia is a non-conventional yeast genus that has gained popularity in synthetic biotechnology due to its desirable characteristics for metabolic engineering and gene editing. The use of Pichia for biotechnological applications has been well established over the past few decades, and its use in gene editing has seen significant progress in recent years.
When compared to the model yeast S. cerevisiae, Pichia has more potent and tightly controlled promoters for high-level recombinant protein expression.[1] As such, P. pastoris has been used as an expression host for synthesizing recombinant proteins for the past 20 years.[2] It is able to perform post-translational modifications without hyperglycosylation and secrete expressed proteins into the cultivation medium. The yeast can be grown at high cell densities on simple media, and heterologous act expression has also been reported.
The development of CRISPR/Cas9 technology has allowed for more efficient and precise gene editing in Pichia, and various modifications to the system have been made to improve its efficiency and specificity. Additionally, other gene editing tools such as TALENs, ZFNs, piggyBac and Cas-CLOVER have all been adapted for use in Pichia. These advancements in gene editing tools have allowed for the development of Pichia strains with improved metabolic pathways for the production of various products such as biofuels, chemicals, and pharmaceuticals.
Current Advancements in Gene Editing with Pichia
Figure 2: Summary of synthetic biotechnology tools and applications.
Pichia is mainly gaining popularity as a model organism for industrial research primarily due to its potential as a cell factory for the biosynthesis of valuable bioproducts. Yeast cell factories have the potential to produce a wide range of bulk chemicals, value-added compounds, and proteins. The fields of synthetic biology and metabolic engineering have both generated great interest in non-conventional yeast species, including P. pastoris and P. kudriavzevii, for industrial production purposes. Synthetic biology is a rational approach that involves studying unique genetic parts or modules from a system and integrating them into a strategically integrative method for potential microbe formation as cell factories.
Pichia pastoris is a preferred host for recombinant protein production due to its ability to produce complex recombinant proteins with mammalian glycosylation profiles. Compared to other simple hosts, such as E. coli and S. cerevisiae, P. pastoris requires simpler media and shorter processing times and has efficient protein secretion mechanisms. Its well-characterized constitutive and inducible promoters, and extensively engineered glycosylation pathways further support its use in recombinant protein production. Its ability to produce membrane proteins with high efficiency and high biomass density is an added advantage.
In recent years, significant advancements have been made in Pichia species for metabolic engineering and the production of specific products. However, further research is necessary to fully understand the physiology and genetics of Pichia. To achieve this, “multi-omics” data should be collected, genetically engineered models created, and the flux balance of the major metabolic pathways quantitatively assessed.
Identifying metabolic bottlenecks, enhancing metabolic fluxes, and maximizing theoretical yields are all crucial steps in building efficient pathways. Additionally, it is important for us to further explore novel enzymes, create proteins with unique functions, and leverage gene editing platforms that offer superior copy numbers, enhanced efficiencies, and better scalability compared to CRISPR/Cas9. Examples of such tools include the piggyBac and Cas-CLOVER gene editing platform.
Improve Your Pichia study Results with PiggyBac and Cas-CLOVER
Figure 3: Demeetra’s flagship Cas-CLOVER gene editing technology
CRISPR/Cas9 technology has been successfully used in many cell lines. However, there are two drawbacks to using CRISPR. The first is the potential for off-target mutagenesis. The second is the entanglements that arise from intellectual property (IP) issues, or a lack of clear commercial freedom to operate, which have limited its commercial use.
Cas-CLOVER, in contrast to Cas9, can target multiple rounds at a specific locus, resulting in a higher frequency of indel without the risk of unintended off-target mutations. Cas9 is prone to causing off-target mutations, even if the single gRNA has multiple base-pair mismatches. The combination of Cas-CLOVER and piggyBac gene editing technology can be utilized for yeast strain engineering to improve commercial manufacturing and product discovery. If you have faced issues like unintended off-target mutations or licensing and operational limitations while using CRISPR for gene editing, Cas-CLOVER is a more efficient alternative. Learn more about our proven gene integration and expression in yeasts here.
References:
- Akansha Shrivastava, Mamta Pal, Rakesh Kumar Sharma. “Pichia as yeast cell factory for production of industrially important bio-products: Current trends, challenges, and future prospects.” Journal of Bioresources and Bioproducts, 2023, doi: 10.1016/j.jobab.2023.01.007.
- Zhu, Taicheng, et al. “Pichia pastoris as a versatile cell factory for the production of industrial enzymes and chemicals: current status and future perspectives.” Biotechnology Journal 14.6, 2019, doi: 10.1002/biot.20180