Gülşah AkçadağA., Elif AltayE.
Pichia pastoris has been described as Zygosaccharomyces pastoris by Alexandre Guilliermond who is French mycologist in 19201. After, similar strains were isolated from oak trees by Herman Phaff and given the name P. pastoris 30-40 years later. Nevertheless, a reclassification occurred in 1995, relocating P. pastoris to a distinct genus identified as Komagataella1,2.
P. pastoris is one of the organisms used as a model organism in molecular biology studies, and there are different important reasons for this. At the beginning, bioproducts which are vaccines, insulin, proteins, and antibodies can be produced on a large scale with low cost since it has high cell density capacity, rapid expression and practical downstream process3,4. Moreover, the genome of P. pastoris is fully sequenced and it is easy to manipulate genetically5. Microbial contamination risk is less with P. pastoris thanks to using methanol as a carbon source. In addition to this information, P. pastoris has post-translational modification which is an essential property in case of heterologous protein production. Therefore, P. pastoris is one of the most predominant yeast species in the case of heterologous protein production in laboratory and industrial scales according to the results of literature studies6.
Pichia pastoris Genetic Background
P. pastoris strains are grouped into two different species as Komagataella pastoris and K. phaffii. However, P. pastoris name is preferred for both species since various strains are under the Komagataella genus1. P. pastoris is a methylotrophic yeast which means it can utilize methanol as a carbon and energy source6. In addition, methanol utilization capacity based on alcohol oxidase genes (AOX1 and AOX2) of P. pastoris determines phenotype7. If P. pastoris has both AOX1 and AOX2 genes, then it is called wild type strains (Mut+) and it can grow on methanol. Mut-strains cannot utilize methanol since it does not contain AOX1 and AOX2 genes. Additionally, there is a MutS phenotype that can grow on methanol slowly since one or more alcohol oxidase genes are deleted6. K. pastoris which is a Mut+ and its ATCC number is 28485 has 4 chromosomes7,8. In addition to this information, its genome size is 9.6 Mb, and it can code 5241 genes with 41.5% GC content9,10.
Different strains of P. pastoris and their features are summarized in Table 1.
| Strain | Genotype | Phenotype | Protease Deficiency |
| X-33 | WT | Mut+ | – |
| GS115 | his4 | Mut+, His- | – |
| KM71 | ∆aox1::SARG4 his4 arg4 | Muts, His- | + |
| SMD1165 | His4 prb1 | Mut+His–Prot–(B–) | + |
| SMD1168 | his4, ura3, pep4::URA3 | (A–, BS, CarbY–) | + |
Pichia pastoris as An Expression System
P. pastoris has various advantages as an expression system. There are strong promoters as Glyceraldehydes-3-phosphate dehydrogenase (GAP), formaldehyde dehydrogenase (FLD), and peroxisomal matrix protein (PEX8) to use in the P. pastoris expression system6,11,12. Most importantly, it can express genes under the AOX1 promoter which is one of the strongest and closely regulated eukaryotic promoters according to literature studies13. Methanol can be used as an inducer in transcription owing to the AOX1 promoter. Various protein yields reach 14.8 g/L under the expression AOX1 promoter14. Moreover, dry weight reaches 200 g/L in the case of acetic acid and ethanol production15.
Figure 2. Methanol utilization pathway of P. pastoris16.
Figure 2 illustrates the crucial roles of AOX1 and AOX2 in the oxidation of methanol to formaldehyde. Subsequently, formaldehyde becomes a key player in either the assimilatory or dissimilatory pathways. When formaldehyde participates in assimilatory pathways, it leads to the formation of glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone (DHA), contributing to biomass generation through various reactions. On the other hand, if formaldehyde is directed towards the dissimilation pathway, each mole of methanol yields 2 moles of nicotinamide adenine dinucleotide hydrogen (NADH), accompanied by the release of CO2 through formaldehyde oxidation16,17.
Moreover, AOX1 promoter can be repressed through various carbon sources such as glycerol and glucose17. Therefore, high biomass can be obtained since the initial carbon source prevents transcription of the gene. Instead of these advantages, AOX1 promoter usage can be harmful on an industrial scale, since methanol can cause toxic effects and fire6. Remarkably, AOX1 promoter is one of the strongest promoters in the case of heterologous gene expression in literature18.
There are synthetic and drug-resistance markers in the P. pastoris expression system. As an example, HIS4, ARG4 and URA3 are synthetic markers while kanamycin and zeocin are drug-resistances19. However, these markers have their own features, and the production process must be planned according to them.
P. pastoris secretion capacity is an essential point in the case of heterologous protein production. P. pastoris has Saccharomyces cerevisiae α-mating factor prepro-peptide, and it is the most frequently used secretion signal leader in the case of P. pastoris20. Indeed, purification steps will be easier and cheaper with P. pastoris expression system thanks to having secretion signal peptide and not having many native secreted proteins6.
Plasmid durability is another important point in the case of expression systems. Plasmids can lose cell division in time. Therefore, it is a bottleneck in the case of heterologous protein productions. P. pastoris expression system has an essential feature, which is called genome integration. Genome integration is an outstanding property owing to sustainability, and it can be achieved through three main protocols electroporating, spheroplast formation, and lithium chloride treatment21. There are different regions for this purpose on P. pastoris chromosome as HIS4 and AOX1 locus. However, AOX1 locus is one of the most chosen loci in the case of homologous recombination21,22. For this purpose, AOX1 locus is preferred frequently since its expression level is high and can be tightly regulated through methanol induction23. In addition, this phenomenon is used as an expression cassette and provides various heterologous protein production.
Figure 3. Genome Integration in P. pastoris21. A. Gene insertion at HIS4 B. Gene insertion at AOX1 C. Gene replacement at AOX1.
P. pastoris has valuable advantages over other expression systems such as bacterial or mammalian. Post-translational modifications, which are most importantly glycosylation, phosphorylation, and acetylation are important in the case of heterologous protein production23.
These modifications can be achieved through mammalian expression systems while it cannot fully perform in the case of bacterial expression systems. Therefore, misfolded, and insoluble proteins are produced in bacterial expression systems21,24. P. pastoris expression system provides post-translational modifications. Indeed, desired protein production is achieved with correct folding20. In contrast to the bacterial expression system, secretion signal capacity is a precisive property for P. pastoris system. In mammalian expression systems, post-translational modifications can be done correctly but the system is more time consuming than yeast expression systems owing to long doubling time. Moreover, mammalian expression systems are more expensive than bacterial and yeast systems because of complex growth medium23.
Usage Areas of Pichia pastoris
P. pastoris is frequently used in laboratory and industrial scales owing to its high concentration yield. There are enormous biomolecules that are produced through this expression system24.
Industrial Enzymes
According to literature studies’ results, biomolecules that are produced P. pastoris expression system are used in different industries.
As an example, wastewater treatment is an essential topic nowadays. Ligninolytic enzymes are mostly preferred in the case of wastewater treatment. Since P. pastoris does not produce cellulolytic enzymes in contrast to other filamentous fungi, produced ligninolytic enzymes can be applied to related industries without extra purification steps. Additionally, different sources of laccase genes that encode ligninolytic enzymes are successfully produced with high activity yield in P. pastoris expression system24-26.
Moreover, there are a lot of essential enzymes that are used in different industry areas such as paper, textile, detergent, food and beverage, wine, and pharmaceuticals. Cellulases, xylanases, proteases, esterases, lipases, amylases, phosphatases, and pectinase are the most frequently used industrial enzymes 25,26. Most of these enzymes are produced through the P. pastoris expression system as shown in Table 2. According to Table 2, fed-batch fermentation is preferred to produce these enzymes, and yield ranges vary due to activity calculations27-34.
Table 2. Bioproducts which are Produced Through P. pastoris Expression System27-34.
| Product Name | Pichia species/strain | Fermentation Type | Titer/Yield | Reference |
| Laccase | P. pastoris | Fed-batch | 0.495 g/L | 27 |
| Lipase | P. pastoris | Fed-batch | 2 g/L | 28 |
| Phytase | P. pastoris | Fed-batch | 5 g/L | 29 |
| Cellulase | P. pastoris | Fed-batch | 2.75 g/L | 30 |
| Transglutaminase | GS115 | Batch | 0.70 U/mL | 31 |
| Pectate lyase | P. pastoris | Fed-batch | 9.5 g/L | 32 |
| α-Amylase | X-33 | Fed-batch | 750 U/mL | 33 |
| Trypsin | GS115 | Fed-batch | 177.85 U/mL | 34 |
Pectinase occurs in three main pectinolytic enzymes as polygalacturonase, pectin methyl esterase, and pectin lyase, and these enzymes have a role in the degradation of pectin according to their reactions25,35. Pectinolytic enzymes are used in different industries such as fruit juice since they can decrease viscosity, remove gel structure, and increase the stability of fruit juice36. Various results are related to producing pectinases through the P. pastoris expression system. For instance, high-level expression of Talaromyces celluolyticus endo-polygalacturonase is achieved through P. pastoris in 201937. In the study, five copies of the gene of interest are integrated into P. pastoris genome, and enzyme activity is reached 7124.8 U/mL with fed-batch cultivation37.
Biofuels
Biofuels are a hot topic worldwide due to the limited energy sources and environmental pollution. In this correction, alternative energy sources studies are increasing day by day. Various biofuels which are biodiesel and bioethanol are produced through the P. pastoris expression system. According to the literature, separate hydrolysis and fermentation techniques are used to get bioethanol (14.36 g/L) with P. stipites30,38. In addition to these, there is another study that is related to bioethanol production (37 g/L) through P. fermentas batch fermentation30,39. Moreover, there is a study which is related to the co-culture method. In this study, recombinant strains of P. pastoris and Saccharomyces cerevisiae are used to produce bioethanol. In this study, 32 g/L bioethanol production is achieved, and this concentration corresponds to %82 of the theoretical yield according to study results40.
Biopharmaceuticals
P. pastoris is frequently used in the biopharmaceutical field to produce diverse products, including vaccines, hormones, cytokines, and antibodies3,4. Notably, insulin, a pivotal hormone with significant implications for various medical conditions, is synthesized using distinct strains of P. pastoris, such as X33, GS115, and SuperMan541,42,43. Immunoglobulin G, a crucial antibody, can be generated with yields of up to 227 mg/L through the utilization of diverse strains of P. pastoris44,45. Vaccines have remained a perennial subject of interest within the pharmaceutical domain for centuries. In this correction, P. pastoris is used to produce various vaccines such as HBsAg, DENV-3 E and HCV E1E246-49. Cytokines are small proteins that have a role in cell signaling. Their recombinant production is an outstanding topic for the pharmaceutical area. According to literature studies results, interleukin-6 and interleukin-3 are successfully produced through wild-type P. pastoris strains with 280 mg/L and 26 mg/L yields50,51. Moreover, another cytokine, recombinant bovine interferon α1 is produced with GS115 strain of Pichia52.
In conclusion, P. pastoris holds significance as a crucial methylotrophic yeast due to its extensive applications in large-scale industrial biotechnology. It stands out as one of the preferred expression systems owing to its capabilities in high cell density cultivation, cost-effective downstream processes, presence of genomic sequence, genetic manipulability, and facilitation of post-translational modifications.
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