Forget about lab-grown meat and synthetic diamonds. The next big thing in biotechnology might just be…yeast. Specifically, two types of yeast: Pichia pastoris and Pichia kudriavzevii. These tiny fungi are proving to be powerful tools for producing everything from life-saving proteins to valuable chemicals.
Yeast Power: A Tiny Microbe with a Big Impact
Pichia pastoris and Pichia kudriavzevii are not your average yeasts. These methylotrophic fungi are known for their ability to produce large amounts of proteins and other valuable compounds. They’re like tiny factories, churning out essential ingredients for a wide range of applications.
Engineering Yeast for a Better World:
Scientists are constantly working to improve these yeasts, using the tools of synthetic biology to create even more powerful strains. They’re tinkering with genes, designing new promoters, and developing innovative plasmids to optimize protein expression and production.
From Proteins to Chemicals: The Many Applications of Yeast
Pichia pastoris is a superstar when it comes to protein production. It’s been used to create everything from enzymes for industrial applications to soy hemoglobin, a potential alternative to animal-based heme. And Pichia kudriavzevii is proving to be a champion for chemical production, thanks to its ability to tolerate acidic environments. It’s being used to produce valuable compounds like succinic acid and D-lactic acid.
The Future of Yeast: A World of Possibilities
As scientists continue to unlock the potential of these yeasts, we can expect to see even more exciting applications in the future. From new drugs and vaccines to sustainable biofuels and biodegradable plastics, yeast is poised to play a major role in shaping the world of tomorrow.
The Takeaway: Yeast – A Microbe with a Mighty Impact
Don’t underestimate the power of yeast. These tiny fungi are changing the world, one protein, one chemical, and one innovative application at a time. Get ready for a future where yeast is a key player in solving some of the world’s biggest challenges.
Rapid advances in metabolic engineering and synthetic biology have attracted widespread attention recently, prompting people to actively explore the potential of many non-model microorganisms. Among them, Pichia pastoris and Pichia kudriavzevii are two outstanding representatives in the field of Pichia pastoris. With their unique biochemical, metabolic and physiological characteristics, they have shown great advantages in promoting large-scale industrial production. The purpose of this review is to first provide an overview of the synthetic biology components and tools developed for these two species, and then to summarize their widespread use as biomanufacturing hosts in the production of recombinant proteins, bulk chemicals, and natural products. Finally, the article will delve into the challenges and potential strategies to expand its application in the wider biotechnology field.
1Introduction
As the demand for fuel and petrochemical products surges, and food shortages and global warming become increasingly serious problems, scientific researchers are actively engaged in the field of engineering microorganisms to produce various needed products. Model organisms such as Escherichia coli and Saccharomyces cerevisiae have been deeply explored and successfully transformed into cell factories to produce high value-added chemicals and proteins. With the rapid development of next-generation sequencing technology and synthetic biology tools, non-traditional organisms with unique characteristics (such as heat resistance, pressure resistance, the ability to utilize special raw materials, and high protein secretion capabilities) have gradually become ideal hosts for industrial processes.
Among them, Pichia pastoris in the genus Pichia pastoris is particularly eye-catching. As a methanotrophic yeast, it can use methanol as the only carbon source and grow to extremely high cell density ( >100 g/L dry cell weight). Its culture medium is low-cost and simple (containing only methanol or glycerol, biotin, salts and trace elements), and is extremely suitable for large-scale industrial production. In addition, P. pastoris can efficiently express heterologous proteins, whether intracellular or extracellular, while effectively controlling the secretion of endogenous proteins to ensure product purity. Its post-translational modification mechanism is complete, including glycosylation, disulfide bond formation and proteolytic processing, which further improves the biological activity and stability of the product.
As a “generally recognized as safe” ( GRAS ) yeast, P. pastoris has been widely used in the production of industrial enzymes, chemicals, therapeutics (such as vaccines and drugs) and protein-based polymers. In recent years, researchers have also successfully applied it to the production of leghemoglobin ( LegH ), giving plant-based meat products a meat-like flavor.
The genome sequence of P. pastoris has been exhaustively annotated, and multiple genome-scale metabolic models have been constructed, providing a solid foundation for engineering modifications. This review will systematically summarize the current synthetic biology components and tools applicable to P. pastoris , covering promoters, terminators, plasmids, genome editing tools, signal peptides, etc., and also discuss in-depth how to improve protein secretion efficiency and expand the application field of this yeast. Engineering research carried out. At the same time, we will also focus on another member of the genus Pichia pastoris – Pichia kudriavzevii , whose acid-tolerant properties show great potential in the production of high value-added organic acids (such as succinic acid, D- lactic acid and itaconic acid).
2 Synthetic biology components and tools
2.1 Promoter and terminator
Promoter strength and tunability are critical for efficient production. The use of strong promoters is generally preferred because expression levels of heterologous genes are usually low, whereas tunable promoters are desirable when multiple heterologous genes are involved in complex pathways. Commonly used promoters include inducible and constitutive promoters. The former allows genes of interest to be turned on or off at different stages through induction or repression of transcription factors. In practice, the use of inducible promoters can first increase the cell density of a culture and then initiate the production of heterologous proteins. This separation of biomass accumulation and protein production provides significant advantages when accumulated intermediates or products are toxic to the cell. However, the use of inducible promoters requires an additional step in the culture process (e.g., carbon source exchange or compound supplementation), which adds additional costs in large-scale industrial production. In contrast, strong and stable expression of genes mediated through constitutive promoters helps reduce operating costs while increasing yields, a common strategy if constitutive expression does not negatively impact cell growth. Influence.
Inducible promoters are generally identified from specific biochemical pathways, whereas constitutive promoters are often derived from housekeeping genes. In P. pastoris , the two most commonly used promoters are the inducible PAOX1 promoter and the constitutive PGAP promoter. PAOX1 is the promoter of the alcohol oxidase gene AOX1 , which can be induced by methanol, a cheap carbon source. When methanol is used as the carbon source, the expression of alcohol oxidase induced by methanol can be as high as 30% of the total soluble protein content , making it one of the most efficient promoters in the field of protein expression in Pichia pastoris.
About twenty years ago, using the PAOX1 promoter, the synthesis level of heterologous proteins could reach approximately 20 g/L . In order to deeply understand the regulatory mechanism of PAOX1 , the scientific research community has invested a lot of efforts. By dissecting its cis-acting regulatory sequence elements, the researchers were able to engineer the promoter by deleting and duplicating putative transcription factor binding sites to create promoters with strengths ranging from 6% to 160% of wild-type PAOX1 . Various variations.
In addition, more than a dozen transcription factors have been confirmed to be involved in the induction process of PAOX1 . This work not only combined the study of cis-acting elements, but also determined the characteristics of the basal promoter based on deletion analysis, ultimately providing a variety of PAOX1 variants with tunable activity, providing better insights into protein expression in Pichia pastoris. A flexible and efficient tool.
PGAP is the key promoter of the glyceraldehyde -3- phosphate dehydrogenase gene GAP in the glycolysis pathway . It is characterized by sustained constitutive expression, although its activity will show differential changes under different carbon source conditions. Drawing on the engineering strategy of PGAP , we constructed a library of PGAP variants, achieved through mutagenesis, with a wide range of activities, ranging from 0.6% of wild-type PGAP to an astonishing 1960% . Studies have revealed and suggested the potential role of multiple transcription factors in the regulation of PGAP . Although these promoters are still under-researched compared with PAOX1 and PGAP , their potential in regulating multi-gene pathways has attracted much attention. However, the cis-acting regulatory sequences of these promoters and their specific mechanisms are still mostly unknown.
In addition, in addition to the promoters mentioned above, there are also two eye-catching promoters: one is the strong ontological promoter PCAT1 derived from the P. pastoris catalase gene , which can exhibit strong activity through methanol induction , especially the P4 variant of PCAT1 , whose activity even surpasses PAOX1 ; the other is the strong heterologous promoter PMOX from the Hansenula polymorpha methanol oxidase gene , the promoter was completely inactive in xylose and sorbitol environments, but showed significant activity in glucose, glycerol and methanol media.
When working in conjunction with the promoter, the terminator ( tt ) also plays a crucial role in regulating gene expression levels, which is mainly attributed to its regulation of mRNA stability and its profound impact on the subsequent translation process. However, compared with the widespread attention that promoter engineering has received, terminator research seems relatively deserted.
In the classic biological model S. cerevisiae , researchers have thoroughly examined and characterized hundreds of terminators in detail, and these terminators have been proven to have the ability to finely regulate protein expression levels over a wide range. . In contrast, in P. pastoris , an equally important expression system, research on the impact of terminators started late and is currently limited to a few recently published documents.
In heterologous expression studies in Pichia pastoris, both endogenous terminators and heterologous terminators are widely used. Commonly used endogenous terminators are mostly derived from the methanol utilization pathway (such as AOX1tt ) or housekeeping genes (such as GAPtt ), which provide stable termination signals for the expression of heterologous genes. Heterologous terminators are mostly derived from other yeast species, such as CYC1tt (derived from cytochrome C isoform 1 ), PRM9tt (derived from pheromone-regulated membrane protein 9 ) and VPS13tt (derived from vesicles) of Saccharomyces cerevisiae Protein sorting-related proteins 13 ), Hansenula polymorpha’s MOXtt (derived from methanol oxidase), and Kluyveromyces LAC4tt (derived from β – galactosidase). The introduction of these heterologous terminators adds more regulatory dimensions to the heterologous expression system of Pichia pastoris.
Up to now, research on the effects of different terminators on heterologous expression in Pichia pastoris has been mainly achieved by evaluating the expression levels under the control of PAOX1 and PGAP promoters. This research strategy revealed to us the potential and value of terminators in optimizing heterologous gene expression.
The researchers analyzed 20 terminators, 15 of which originated from the endogenous methanol assimilation pathway and the remaining five from Saccharomyces cerevisiae . Under the regulation of PAOX1 , these terminators can all achieve comparable green fluorescent protein ( GFP ) expression levels, and the lowest active terminator can still reach 57% of the highest active terminator . The researchers focused specifically on terminators derived from highly expressed endogenous genes, including numerous ribosomal terminators. Using Saccharomyces cerevisiae CYC1tt as a reference, all ten terminators produced similar GFP levels under PGAP regulation . Notably, in both studies, heterologous terminators of target genes exhibited comparable or even higher activity than endogenous terminators, suggesting that terminators isolated from other yeasts can be used in Pichia pastoris pastoris ) are effectively identified. Recently, Ito et al. constructed a catalog of 72 terminators, including 28 endogenous terminators, 41 heterologous terminators derived from S. cerevisiae, and three strong synthetic terminators originally developed for S. cerevisiae. Under the regulation of PGAP , these terminators achieved 17- fold tunability in Pichia pastoris . In this study, AOX1tt appeared to produce the highest activity regardless of which promoter was used.
A recent study used Candida antarctica lipase B ( CALB ) as a reporter protein and revealed the close correlation between terminator activity and promoter activity. The study compared ten terminators derived from the endogenous methanol utilization pathway, glycolysis, tricarboxylic acid ( TCA ) cycle and other housekeeping genes, as well as five terminators from Saccharomyces cerevisiae. Its activity was evaluated by measuring the corresponding CALB activity under the control of PAOX1 and PGAP . It was found that having three terminators resulted in lower lipase activity when paired with PAOX1 but higher activity when paired with PGAP compared to AOX1tt , suggesting that terminator performance is not independent of promoter effects. , may also be affected by regulatory mechanisms observed in promoter studies. However, in P. pastoris , the mechanism of how each terminator enhances expression under the action of different promoters is unclear. Furthermore, the terminator of dihydroxyacetone synthase ( DHAStt ) provided slightly higher CALB expression levels than AOX1tt under PAOX1 control , but was almost three times more active under PGAP control. Therefore, DHAStt can serve as a potential strong terminator when seeking high heterologous expression in P. pastoris . In summary, terminators play a key role in protein expression, but the regulatory mechanisms mediated by them still require further study to elucidate.
2.2 Cell-free plasmids and integrated plasmids
In P. pastoris , most protein expression and metabolic engineering tasks are achieved through genome integration. This includes large-scale relocation of integration sites, untargeted integration that may affect cell growth, and co-integration of DNA elements from the F plasmid used to prepare the shuttle plasmid and the E. coli host genome . Therefore, tedious screening procedures must be employed to identify transformants with target integration.
Another strategy involves the use of replicative plasmids, which have high transformation efficiencies and facilitate screening, although their stability sometimes poses a problem. Autonomous replicating sequences ( ARS ) are key components in replicating plasmids. The first P. pastoris- specific ARS , PARS1 , was discovered 35 years ago, which enabled the use of replicative plasmids with high transformation efficiency. Over the past decade, other elements that serve as alternative ARSs in P. pastoris have been discovered, such as the 452 bp panARS identified from K. lactis and the 1442 bp mitochondrial DNA fragment identified from P. pastoris itself . However, plasmids using these ARS exhibit poor stability during mitotic isolation, which is detrimental for industrial applications. Recent studies have shown that this inherent instability results from the lack of a centromere ( CEN ), another genomic element that directs stable chromosome segregation and, therefore, can be used to increase plasmid stability during cell division.
Centromere sequences ( CENs ) are DNA sequences recognized by the centromere complex and subsequently interact with spindle microtubules to ensure equal distribution of chromosomes between two dividing cells during mitosis and meiosis. In S. cerevisiae , a 125 bp CEN with an ARS is widely used in all low-copy plasmid vectors. Similar to ARS , CENs are also species-specific, and recent studies have identified four putative CENs , corresponding to the four in P. pastoris . In a more recent study, a new autonomously replicating plasmid containing the entire putative centromeric region from chromosome 2 ( Cen2 ) was constructed. This plasmid can replicate and be stably distributed in Pichia pastoris. In this Cen2 , a sequence of approximately 111 bp was found that can achieve autonomous replication and can serve as a new ARS . Another study confirmed that entire CENs from chromosomes 1 and 4 ( Cen1 and Cen4 ) can confer plasmid replication stability, although in another study Cen4 did not support high numbers of transformants. Although these new plasmids exhibit relatively high stability and have the potential to accelerate cloning and high-throughput screening, inclusion of the entire Cen sequence may lead to other undesirable results. First, such plasmids can only be maintained at low copy numbers because of their chromosome-like segregation mechanism, in contrast to plasmids using PARS1 . Furthermore, since all Cen sequences are more than 6 kb in length , the plasmid size will be larger, which is not conducive to transformation, especially when cloning large pathways. Finally, having the entire centromeric sequence may lead to unexpected genomic integration or DNA exchange with chromosomes, which increases the difficulty of screening.
Therefore, until the functional mechanisms of ARS and CEN are clearly elucidated and the sequences are optimized in P. pastoris , genome integration is considered an alternative strategy for the expression of heterologous genes and pathways. Genomic integration is usually achieved via single or double crossover. Single crossover integration requires that the circular vector contains the same sequence as the target site in the P. pastoris genome. After transformation, the linearized vector, including the gene of interest, selectable marker, and backbone, is inserted into the target site, forming two copies of the target site sandwiching the inserted vector. The PAOX1 region and the auxotrophic gene HIS4 have been widely used as target sites for single-crossover-mediated integration, with integration efficiencies of 50-80% . However, this mode of integration may introduce elements outside the expression cassette, such as those responsible for replication of the shuttle vector in E. coli . Furthermore, a second single crossover may reoccur when selection pressure is removed, especially on the genome between two identical target sequences, which may result in the loss of the integrated expression cassette. To achieve double-crossover integration, an expression cassette containing a selectable marker is typically transformed with arms homologous to the target site, resulting in the genomic sequence between the two homologous sequences being directly replaced by the expression cassette. Surrounding the gene of interest and the selectable marker with 5′ and 3′ AOX1 sequences results in disruption of AOX1 , thereby altering the phenotypic matrix utilization of P. pastoris . Finally, selectable markers for screening typically include auxotrophic genes such as HIS4 , URA3 , and ADE1 , as well as genes encoding resistance to zeocin and G418 sulfate.
2.3 Gene editing and integration sites
Clustered regularly interspaced short palindromic repeats ( CRISPR ) technology has become a revolutionary tool to achieve genome integration in P. pastoris . The principle is that the Cas9 protein, with the assistance of guide RNA ( gRNA ) molecules, can target any sequence containing 2 Accessible loci separated by -6 bp from adjacent motifs. However, studies have pointed out that the Cas9/gRNA complex may cause toxic effects due to off-target effects, and therefore, the expression level of Cas9 must be appropriately controlled to ensure the required efficiency. For example, using a weaker promoter for Cas9 expression can improve transformation efficiency and growth rate. Cas9 cleaves the genome and introduces double-stranded breaks ( DSBs ), which can be repaired by the nonhomologous end-joining ( NHEJ ) mechanism in the absence of donor DNA , resulting in insertion and deletion ( indel ) mutations at the target locus . In P. pastoris , due to its inherent strong NHEJ activity, multi-locus disruption or deletion can be efficiently achieved by co-transforming different gRNAs . For example, researchers successfully achieved simultaneous mutation of GUT1 and AOX1 by expressing two gRNAs on CRISPR/Cas9 plasmids . By testing different gRNA combinations, the highest dual editing efficiency of 69% was obtained . However, active NHEJ machinery is an obstacle to achieving precise locus-specific integration via homologous recombination ( HR ). When donor DNA is provided , HR must compete with primary NHEJ to repair DSBs , and under selective pressure, markers can be randomly integrated through NHEJ . Wild-type P. HR activity in pastoris is naturally much lower than in NHEJ , resulting in many false positives. Therefore, achieving precise integration in P. pastoris is much more difficult than in other yeast species where HR is dominant, such as S. cerevisiae . This limitation can be overcome by deleting the KU70 and KU80 genes that encode the two proteins that form the heterodimer . In a recent study, it was reported that NHEJ was largely inhibited upon knockdown of KU70, thereby enhancing the role of HR in repairing DSBs and increasing target integration efficiency to approximately 100% .
While inhibiting the non-homologous end joining ( NHEJ ) mechanism, future research should focus on improving the efficiency of homologous recombination ( HR ) to enhance the gene editing ability of CRISPR/Cas9 technology in P. pastoris . For example, strategies to overexpress RAD family recombinases could be explored . In S. cerevisiae , overexpression of Rad51 has been shown to improve the accuracy of gene integration, while overexpression of an engineered Rad51 variant with higher affinity for the recombinase Rad54 significantly improved the accuracy of gene integration. Improved targeting efficiency in Saccharomyces cerevisiae. In addition, expressing Rad52 from Saccharomyces cerevisiae in Yarrowia lipolytica has been shown to increase targeting efficiency from 15% to 95% . When the KU70 gene is disrupted, certain chemicals such as hydroxyurea are used to synchronize Yarrowia lipolytica cells to the S phase of the cell cycle, the period when HR activity is highest. Similar strategies can be used to enhance HR efficiency in Pichia pastoris .
Theoretically, any site on the genome can serve as a potential integration target, except for sites associated with essential genes. However, several additional characteristics directly affect integration efficiency. First, higher accessibility gives the Cas9/gRNA complex, and subsequently the donor DNA, an increased chance of forming complexes with the genome during cleavage and repair processes, which is particularly important for the integration of large pathways. AOX1 , DAS1 , DAS2 , and GUT1 are widely targeted because of their easy accessibility. Furthermore, the nanoenvironment surrounding the integration site is critical for determining the expression levels and dynamics of the integrated gene. Although high expression levels do not necessarily lead to high yields, it is often preferred by the rate-limiting step of synthetic products in multistep pathways.
Increasing the copy number of specific genes has proven to be an effective strategy to enhance heterologous protein production. In view of this, the gene locus encoding ribosomal RNA ( rRNA ), namely ribosomal DNA ( rDNA ), has been widely adopted due to its repetitive sequence properties. By designing plasmids containing a single gRNA , multiple rDNA sites can be targeted simultaneously. In P. pastoris , each rDNA repeat unit is composed of 25S , 5.8S , and 18S rRNA genes, which are arranged in a head-to-tail tandem manner, and there is a non-transcribed spacer ( NTS ) between the two rDNA repeat units. . These rDNA repeats and their location on chromosomes have been described in detail in many yeast species for decades. For example, researchers have successfully used CRISPR/Cas9 technology to integrate ten copies of the resveratrol biosynthetic pathway from Herpetosiphon aurantiacus , Arabidopsis thaliana , and Vitis vinifera into the NTS region of O. polymorpha without using a selectable marker . In Y. lipolytica , Luu et al. obtained eight copies of the integrant of the red-spotted grouper neuronecrosis virus capsid protein at the 26S rRNA site through homologous recombination ( HR ) . In P. pastoris , high copy number integration of human serum albumin and human superoxide dismutase was achieved by targeting the NTS region of the rDNA locus and repeating selection at increasing concentrations of zeocine ™.In addition to expressing the protein as a product, this strategy has also been applied to integrate the D- lactate dehydrogenase gene ( D-LDH ) into the rDNA locus and achieve copy number amplification through gradually increasing antibiotic concentrations, thereby enhancing P. pastoris Production of D- lactic acid.
2.4 Common strains
Y-11430 ( CBS-7435 ), GS115 and X-33 are commonly used P. pastoris strains. Y-11430 is a wild-type strain originally isolated from California black oak and deposited in the United States Department of Agriculture Cultural Collection ( USDA-NRRL ). It is known for its robust growth rate and high activity in the methanol utilization pathway. GS115 is a histidine auxotrophic strain obtained by mutagenesis of Y-11430 with nitrosoguanidine . Due to its ease of integration and screening, HIS4 has become very popular when used as a selection marker. X-33 is a revertant obtained by complementing the HIS4 gene of GS115 , and zeocin or blasticidin can still be used to select X-33 transformants carrying antibiotic resistance genes . X-33 shares some mutations with GS115 in genes encoding cell wall biosynthesis that enhance secretion of membrane-associated proteins and lead to higher transformation efficiency than other species with thick cell walls, making X-33 a heterologous protein Expressed in popular commercial strains.
2.5 Signal peptide mediates protein secretion
Protein secretion is a complex process that involves multiple steps that work together to produce mature, active proteins. The signal peptide is a short amino acid sequence, usually located at the N- terminus of a newly synthesized polypeptide chain, and is responsible for guiding the protein into the secretory pathway. In P. pastoris , the most commonly used signal peptide for recombinant protein expression is derived from the α – mating factor (α -MF ) propeptide of S. cerevisiae (see Figure 1 ). The α -MF propeptide consists of a pre-signal sequence containing 19 amino acids and a pre-sequence containing 66 amino acids. Propeptides usually contain three regions: a positively charged N- terminal region, a central hydrophobic region, and a polar C- terminal region. Processing of α -MF secretion signals involves three main steps. First, a signal peptidase in the endoplasmic reticulum ( ER ) cleaves the pre-signal sequence. Next, the Kex2 endopeptidase in the Golgi apparatus cleaves the leader sequence at the dibasic KR site. Finally, the Ste13 protein removes the EA repeats. α -MF has been used in Pichia pastoris to produce a variety of recombinant proteins, such as endoglucanase III from Trichoderma viride , human P53 protein, and manganese superoxide dismutase ( PoMn SOD ) from Lentinus edodes. Many recombinant proteins have been successfully expressed in Pichia pastoris using their native signal peptides. For example, the activity of Aspergillus oryzae alkaline protease with its natural signal peptide is 1.5 times higher than that using α -MF secretion signal peptide ; while the laccase activity of the white rot fungus Polypore TR16 with its natural signal peptide is Three times higher activity than using α -MF secretion signal peptide.
In order to improve the efficiency of α -MF signal sequences, various strategies have been implemented, including codon optimization, error-prone PCR mutagenesis, deletion mutagenesis, and synthetic signal peptides. After codon optimization, the production of phytase increased by about seven times, while the production of lipase CALB increased by 132-295% compared with before optimization . Through error-prone PCR and library screening, an α -MF mutant increased the secretion of single-chain antibody 4m5.3 to 16 times that of the wild type. This improvement was also seen in the production of other single-chain antibody fragments as well as two structurally unrelated proteins, interleukin -2 ( IL-2 ) and horseradish peroxidase ( HRP ). In a recent study in S. cerevisiae , the α- 9H2 – guided sequence and remove potentially harmful or neutral mutations), an optimized α -MF was obtained , named α OPT , with four mutations ( A α 9D , A α 20T , L α 42S and D α 83E ). Compared with α -MF , α OPT can increase the secretion of two laccases, PK2 and ApL, by approximately 14- fold and 26- fold, respectively. Combinatorial saturation mutagenesis at positions 86 and 87 of the α OPT leader sequence can further enhance laccase secretion. Apply this alpha OPT to P. Protein expression in pastoris is attractive. Furthermore, deleting 57-70 amino acid residues in the α -MF propeptide increased HRP activity by more than 50% and approximately doubled CALB activity compared with the wild-type α -MF signal sequence . In another study, this approach resulted in increased production of granulocyte colony-stimulating factor ( G-CSF ) to 39.4 ± 1.4 mg/L . Structural studies show that a specific orientation is required between the N- terminus and C -terminus of α -MF propeptide to interact with the secretion machinery and thereby promote protein secretion. Mutations created near these termini often negatively affect secretion, whereas changes within the propeptide may favor secretion if these mutations stabilize the N- and C- termini. By combining the established leader sequence and the deletion of α -MF , Obst et al. designed several synthetic secretion signal peptides and used red fluorescent protein ( RFP ) and yeast enhanced green fluorescent protein ( yEGFP ) as reporter genes in different Characterized under the promoter. However, although the secretion efficiency of these synthetic hybrid peptides varied more than tenfold, all were less efficient than α -MF except αMF_no_EAEA under certain promoters . Fusion of the S. cerevisiae Ost1 signal sequence and the α -MF proregion, with two mutations, increases the secretion of far-red fluorescent protein E2-Crimson by 20- fold and the lipase BTL2 by ten-fold.
In addition to modifications of α -MF , potential secretion signal peptides can be identified by computational analysis and further confirmed experimentally. The researchers used five computer programs, SignalP4.1 , Phobius , WolfPsort0.2 , ProP1.0 and NetNGlyc1.0 , to identify eight signal peptides from the sequences of 56 endogenous and exogenous proteins . D scores were higher than S. cerevisiae α -MF . Among the eight signal peptides, five ( SP13 , SP23 , SP24 , SP26 , and SP34 ) with D scores higher than 0.8 were selected to study their efficiency in secreting recombinant human growth hormone. SP23 has the highest secretion efficiency. There are also eight commercially available signal peptides ( PichiaPink ™ Secretion Signal Set) used for protein expression in P. pastoris .
2.6 Co-expression of molecular chaperones to promote protein folding
Secreted proteins are transported into the endoplasmic reticulum in an unfolded state and subsequently fold into their native conformation with the assistance of molecular chaperones. Only after the protein is folded correctly will it be transported from the endoplasmic reticulum to the Golgi apparatus for further modification and ultimately to its target location within or outside the cell. The folding process of secreted proteins is complex and error-prone. Once unfolded proteins accumulate in the endoplasmic reticulum, the unfolded protein response is triggered, aiming to reduce the number of newly unfolded proteins entering the endoplasmic reticulum and enhance the folding capacity of the endoplasmic reticulum. If the endoplasmic reticulum is overloaded with too many misfolded proteins, cells will initiate an apoptotic process. In addition, misfolded proteins can also be transported from the endoplasmic reticulum to the cytoplasm, undergo ubiquitination, and be degraded by the proteasome. This process is called endoplasmic reticulum-associated degradation ( ERAD ). In order to improve the production efficiency of recombinant proteins in Pichia pastoris, endogenous or exogenous molecular chaperones can be overexpressed to promote the correct folding and secretion of proteins. Molecular chaperones mainly include two categories: molecular chaperones and chaperone proteins. The former binds to short fragments of substrate proteins, while the latter forms a barrel-like folding cavity to sequester all or part of the unfolded protein and ensure its correct folding.
Protein disulfide isomerase ( Pdi ) is a chaperone protein widely used in the endoplasmic reticulum lumen. Its main function is to catalyze the formation and isomerization process of disulfide bonds, that is, to transform the incorrectly connected protein structure into the correct one. disulfide bond connection form and assist the protein to achieve the correct folding state. When Pdi was co-expressed with IL-1 receptor antagonist and human serum albumin fusion protein ( IH ), the production of IH was significantly improved compared to strains expressing only high copies of IH protein. Other studies have shown that E. coli AppA phytase contains an additional discontinuous disulfide bond. When co-expressed with Pdi , the thermal stability of phytase ApV1 is enhanced. Therefore, compared with expressing ApV1 alone , its yield is higher. increased by approximately 12 times.
Immunoglobulin-binding protein ( BiP ) is another chaperone protein that is abundant in the endoplasmic reticulum. As an important member of the heat shock protein Hsp70 family, BiP not only promotes the folding process of proteins, but also causes endoplasmic reticulum-associated degradation ( ERAD) . ) pathway plays a key role. In Pichia pastoris, when BiP was co-expressed with the A33 single-chain antibody fragment ( A33scFv ), the folding capacity of the endoplasmic reticulum was enhanced, resulting in an approximately threefold increase in the secretion of A33scFv . In addition, co-expression of the partner gene KAR2 with different copy numbers of the gene encoding hydrophobin ( HFBI ) also led to an increase in HFBI secretion. Among them, the highest secretion amount of 3 copies of HFBI was compared with the strain that only overexpressed a single copy of HFBI . 22 ± 1.6 times higher .
In addition to chaperones, chaperone proteins have also been engineered to promote protein production in P. pastoris . D- phenylglycine aminotransferase ( D-PhgAT ) derived from Pseudomonas aeruginosa ST-201 is an intracellular protein that is difficult to express in a soluble active form. The researchers overexpressed the enzyme in Pichia pastoris and observed that most of the D-PhgAT protein was insoluble. By co-expressing D-PhgAT with the E. coli molecular chaperone protein GroEL-GroES , a large amount of soluble D-PhgAT was successfully produced , and its activity was significantly improved. Compared with the D-PhgAT gene expressed alone , when ten copies of the molecular chaperone were co-expressed, the volume activity increased 14,400 times. In another study, GroEL-GroES located in the endoplasmic reticulum was co-expressed with extracellular bacterial phytase or intracellular D-PhgAT in Pichia pastoris. The volume activity of extracellular phytase increased by 1.5 to 2.3 times compared to when phytase was expressed alone. However, most of the D-PhgAT protein is still inactive and exists in the insoluble protein fraction. These results suggest that the GroEL-GroES chaperones, when located in the same compartment, may potentially promote functional protein production in Pichia pastoris.
2.7 Cell surface display
Cell surface display technology is a promising method that achieves expression and engineering on the cell surface by fusing functional proteins with anchoring proteins. The technology has a wide range of applications, including but not limited to: serving as a whole-cell biocatalyst, participating in biosorption and bioremediation, designing biosensors, developing vaccines and antibodies, performing epitope mapping, library screening, and protein engineering. The anchoring protein can be fused to the target protein at the N -terminus or C- terminus. The fusion sequence between the target protein and the anchoring protein, as well as the choice of linker, will have an impact on display efficiency and functional properties.
In Pichia pastoris, commonly used anchoring proteins include Aga1 , Sed1 , Tip1 , Aga2 and Flo1 , which are derived from Saccharomyces cerevisiae, as well as Pir1 and Pir2, which are unique to Pichia pastoris. In one study, by screening the genome of Pichia pastoris GS115 , researchers discovered 13 endogenous glycosylphosphatidylinositol-modified cell wall proteins, three of which were selected as anchor proteins for displaying lipase CALB . These three anchoring proteins (i.e., GCW21 , GCW51 , and GCW61 ) were also used to display bacterial PETase on the surface of Pichia pastoris to degrade highly crystalline polyethylene terephthalate ( PET ). The turnover rate of whole-cell biocatalysts displaying PETase was approximately 36 times higher than that of purified PETase . In addition, another anchoring protein Flo9 was identified in Pichia pastoris. Lipase B displayed using Flo9 showed higher thermal stability at 45 °C and remained stable in organic solvents.
The P. pastoris X-33 strain has been genetically engineered to assemble protein complexes, such as microcellulosomes, on the cell surface. By fusing the truncated CipA protein, which contains a cellulose-binding module and two cohesin modules of Clostridium acetibutyricum, to the C- terminus of the anchored flocculation protein Flo1 of Saccharomyces cerevisiae, and of Nasutitermes takasagoensis Endoglucanase ( NtEG ) fused to the dockerin module successfully achieved this goal. These fusion proteins were expressed in two different P. pastoris X-33 strains and assembled into microcellulosomes through co-culture. By immunofluorescence and Western blotting, the researchers confirmed the successful display and assembly of CipA as well as cohesin and dockerin modules on the cell surface.
Compared with unfixed NtEG , NtEG fixed on microcellulose bodies increased the hydrolysis efficiency of carboxymethyl cellulose ( CMC ), microcrystalline cellulose ( Avicel ) and filter paper by 1.4 times, 2.0 times and 3.2 times, respectively. . In addition, another study utilized the ultrahigh-affinity IM7/CL7 protein to assemble microcellulosomes. IM7 (containing one, two or three units) was fused to the N terminus of the anchoring protein SED1 of Saccharomyces cerevisiae and expressed in P. pastoris . Endoglucanase ( EG ), exoglucanase ( CBH ), beta – glucosidase ( BGL ) and carbohydrate binding module ( CBM ) from Thermobifida fusca , each with an N- terminal CL7 tag fusion, expressed alone in E. coli. Proteins secreted from E. coli cultures are assembled in vitro and displayed on the surface of P. pastoris cells. Display systems with two or three IM7 units hydrolyzed Avicel , phosphate-swollen cellulose ( PASC ) and CMC with comparable or even higher efficiency than free cellulases. When three IM7 units participated in CMC fermentation, the ethanol production reached 5.1 g/L .
In a recent study, researchers fused the envelope of ZIKV (Zika virus) and specific immunogenic epitopes of the NS1 protein, as well as a combination of these two epitopes, to the N- terminus of part of Ag α 1 ( C- terminal portion, nucleotides 970-1950 ) and was displayed on the surface of P. pastoris GS115 . The ability of recombinant yeast to stimulate immune cells was evaluated in vitro by using immune cells isolated from mouse spleens. The results showed that P. pastoris displaying the EnvNS1 epitope showed better effects in producing IL-6 , IL-10 and tumor necrosis factor – alpha ( TNF- alpha) cytokines and increasing CD4+ , CD8+ and CD16+ lymphocytes, compared with ZIKV infection is similar. The discovery of these epitopes will be of great value in the development of vaccines against ZIKV infection.