1、 Background of Pasteur Pichia pastoris Pasteur Pichia pastoris (hereinafter referred to as Pichia pastoris) is a type of methanol tolerant yeast isolated by Herman Phaff from oak trees in 1956, named Pichia pastoris. It was reclassified as Komagataella in 1995 and later subdivided into two species: Komagataella pastoris and Komagataella phaffii. At present, the widely used commercial strains X33 and GS115 of Pichia pastoris belong to K Phaffii species, but most literature and databases still use the old name P pastoris.

The methanol metabolism pathway of Pichia pastoris: methanol is oxidized to formaldehyde and hydrogen peroxide through alcohol oxidase (AOX1/2). Hydrogen peroxide (H2O2) is catalyzed by catalase (CAT) to decompose into water and oxygen, while formaldehyde has two destinations: a portion of formaldehyde enters the assimilation pathway and is assimilated in the dihydroxyacetone synthase (Das1/Das2) and xylulose monophosphate cycle, regenerating 3-ketose-5-phosphate molecules and constructing a glyceraldehyde-3-phosphate as a precursor for biomass formation; The other part enters the pathway of alienation, where formaldehyde is oxidized to formic acid to generate CO2, producing cytoplasmic NADH, which can be used as a reducing equivalent or enter the respiratory chain to provide ATP.

Phenotype of Pichia pastoris: In the expression system of Pichia pastoris, there are two genes encoding alcohol oxidase, namely AOX1 and AOX2. The expression level of AOX2 is much lower than that of AOX1, and the activity of alcohol oxidase in cells is mainly provided by the AOX1 protein expressed by the AOX1 gene. Based on the presence or absence of two alcohol oxidase genes, there are three different methanol utilization (Mut) phenotypes, and the wild-type with active AOX1 and AOX2 is called Mut+; When the AOX1 gene is lost and only AOX2 exists, most of the alcohol oxidase activity is lost. This type of cell has a low ability to utilize methanol and grows slowly on methanol culture medium. The phenotype of the strain is MutS (Methanol Utilization Slow), and MutS yeast strain has a mutated aox1 site, but AOX2 is wild-type; And in strains where both AOX genes are missing, it is called Mut −.

2、 The tools and techniques for expressing recombinant proteins in Pichia pastoris play an important role in the process of recombinant protein production using mature and constantly expanding tools and techniques, enabling P Pastoris becomes an efficient expression system.

2.1 The various strains of Pasteur Pichia pastoris currently used as host strains are all derived from the original Y-11430 strain, and the selection of strains includes wild-type, nutritionally deficient, protease deficient, and sugar engineered strains (Table 1). When proteins are expressed intracellular, MutS phenotype can be prioritized, and both Mut+and MutS can be used for secretion expression. Most yeast strains such as SMD1168, GS115, KM-71 are histidine dehydrogenase deficient and can be screened for recombinants using histidine free media. SMD1168 is a protease deficient type, suitable for situations where it is necessary to protect expressed proteins from endogenous protease degradation, such as unstable proteins or protein complexes.

In yeast expression systems, the plasmids or vectors used for transformation often do not contain the replication source points of yeast itself. If the circular recombinant plasmid is directly transferred into yeast cells, it cannot exist stably. Therefore, linearization treatment must be carried out to integrate it into yeast chromosomes through homologous recombination, so that the target gene can exist stably in yeast cells. The expression vectors of yeast expression system are mainly integrated vectors, which commonly include intracellular protein expression and secreted protein expression. Intracellular expression vectors: mainly including pGAPZ, pPIC3, pPICZ, pPSC3K, pHIL-D2, etc. Expressing the target gene inside the cell can avoid yeast glycosylation and is suitable for cytoplasmic expression or protein expression without glycosylation. The expression level is higher than that of extracellular secretion, but purification is relatively complex. The vectors secreted for extracellular expression include pPIC9, pHIL-S1p, pPICZ α, pYAM75P, etc. Multi copy insertion expression vectors: pPIC9K, pPIC3.5K. Multi copy integration of recombinant genes can increase protein expression levels.

2.3 Promoter Pichia pastoris provides a variety of well characterized constitutive and inducible promoters for selection. Constituent promoters provide simplicity and relatively constant expression levels, while inducible promoters are commonly used when isolation, growth, and production are required, which can prevent fast-growing non recombinant cells or the production of toxic proteins (Table 3). Constituent promoters such as pGAP and pGCW14 are commonly used for large-scale recombinant protein production, but are not suitable for expressing proteins that are toxic to cells. Induced promoters such as pAOX1 are strong inducible promoters that are highly activated in the presence of methanol to efficiently drive the expression of target genes, making them suitable for large-scale production; PFLD1 can induce the expression of recombinant FLD1 protein using methanol or methylamine, and glycerol or glucose can replace methanol. The toxicity of methanol has prompted extensive research on expression systems that do not rely on methanol, such as pGTH1 induction under glucose restriction conditions, which promotes metabolic transformation of cells under specific culture conditions, thereby increasing the yield of target products; PTHI11 is activated at low concentrations of thiamine and regulates gene expression by adjusting the concentration of thiamine; PADH3 drives gene expression in the presence of ethanol as a carbon source.

2.4 Signal peptides and stop signal peptides help locate target proteins to the secretion pathway of cells, ensuring their correct folding and subsequent modification, which is crucial for high-level expression and secretion of functional proteins. The commonly used signal peptides in Pichia pastoris include the α - Mating Factor (α - MF) of brewing yeast, Sucrose Converting Enzyme 2 (SUC2), and Pichia pastoris Acid Phosphatase (PHO1). α - MF is the most commonly used and is mainly used for the secretion and expression of peptides and small proteins. Researchers have discovered four new endogenous signal peptides in the reported protein and genome secreted by Pichia pastoris: Dan 4, Gas 1, Msb 2, and Fre 2, expanding the signal peptide library available for recombinant protein expression and secretion in Pichia pastoris.

Conclusion: Pichia pastoris is the preferred expression platform for recombinant protein production, with advantages such as efficient expression system, high cell density growth, high-level recombinant protein production, diverse post-translational modifications, simplified purification process, and safety without endotoxins and viruses. Pichia pastoris has multiple constitutive and inducible promoters, which can precisely control gene expression under different culture conditions, thereby achieving efficient production of recombinant proteins. However, challenges such as methanol toxicity, protein hydrolysis and truncation, limitations of basic research tools, and insufficient expression levels still need to be overcome.