Pichia Pastoris Research Articles

A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future.

Promoters in Pichia pastoris: A Toolbox for Fine-Tuned Gene Expression.

This chapter reviews the different promoters used to control gene expression in the yeast Pichia pastoris, mainly for recombinant protein production. It covers natural inducible, derepressed, and constitutive promoters, as well as engineered synthetic/hybrid promoters, orthologous promoters from related yeasts, and emerging bidirectional promoters. Key examples, characteristics, and regulatory mechanisms are discussed for each promoter class. Recent efforts in promoter engineering through rational design, mutagenesis, and computational approaches are also highlighted. Looking ahead, we anticipate further developments that will enhance promoter design for Pichia pastoris. Overall, this comprehensive overview underscores the importance of promoter choice and engineering for fully harnessing Pichia pastoris biotechnological potential.

Optimal fermentation conditions for growth and recombinant protein production in Pichia pastoris: Strain selection, ploidy level and carbon source

High-cell-density fermentation is a critical aspect of industrial protein production, requiring the selection of an optimal growth medium and carbon source. Pichia pastoris, a methylotrophic yeast, has been established as a widespread recombinant protein expression system in the food and pharmaceutical industries. The primary objective of this work was to create a superior platform for producing alternative proteins thus contributing to future innovation in these sectors. This study compared three wild-type strains, with two of them also analyzed in their diploid versions, using shake flasks and bioreactors. It investigated glucose and glycerol as carbon sources using mCherry as a protein model. Glycerol emerged as the preferred carbon source, resulting in over 40% increase in biomass concentrations compared to glucose across all strains. Notably, wild-type strain Y-7556 reached an exceptional biomass concentration of 244 g DCW/L in just 48 h, the highest reported to date, highlighting the potential of high-cell-density fermentation in P. pastoris. Regarding protein expression, the diploid version of Y-11430 produced >43% of purified mCherry protein after 123 h of fermentation, compared to the haploid counterpart. Our findings underscore the advantages of diploid strains, optimized fermentation media, and carbon source selection, effectively addressing crucial gaps in the literature.

Improving heterologous expression of laccase by Pichia pastoris via vanillin-induced stress response and its application for removing inhibitors of lignocellulose hydrolysate

Vanillin-sensitive promoters were screened and employed to improve the heterologous expression of laccase in Pichia pastoris. The recombinant yeast could well remove phenolic compounds to improve the fermentability of wheat straw hydrolysate.

Increasing the production of insulin glargine in Pichia pastoris through medium modification

Increasing the production of insulin glargine in <i>Pichia pastoris</i> through medium modification

Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Expression, Purification and Enzymatic Activity of α-1,2 Mannosidase I Derived from Trichoderma reesei in Pichia pastoris

Engineering protein translocation pathway to improve recombinant proteins in Pichia pastoris

Pichia pastoris is one of the most commonly used hosts for producing heterologous proteins, whereas production levels vary depending on the protein of interest and are also regulated by regulatory factors. We conducted RNA-seq by expressing the reporter EGFP and observed significant upregulation of certain subunits (Sec61p, Sbh1p, Sss1p, Sec66p and Sec72p) of the Sec complex in the high-expression recombinant GS115 stains. The overexpression of these genes may increase the expression levels of heterogeneous proteins. In this study, the endogenous promoters of the Sec complex subunits Sbh1p, Sss1p, Sec66p and Sec72p were isolated and verified their activity using the Lac-Z reporter gene. Sss1, Sbh1, Sec66 and Sec72 were overexpressed under the control of their own promoters in Pichia pastoris, respectively. The overexpression of Sss1, Sbh1, Sec66 and Sec72 in cells was confirmed by fluorescent microscope and Western blot analysis. The α-amylase was employed to evaluate the effect of overexpression of the Sec subunits on the heterologous protein expression. The results demonstrated that the α-amylase activity increased by 16%, 58%, 16% and 17% in the strains overexpressing Sss1, Sbh1, Sec66 and Sec72, respectively. Engineering the protein translocation pathway can be an alternative to enhance heterogeneous proteins in Pichia pastoris expression system.

Considerations for Glycoprotein Production.

This chapter is intended to provide insights for researchers aiming to choose an appropriate expression system for the production of recombinant glycoproteins. Producing glycoproteins is complex, as glycosylation patterns are determined by the availability and abundance of specific enzymes rather than a direct genetic blueprint. Furthermore, the cell systems often employed for protein production are evolutionarily distinct, leading to significantly different glycosylation when utilized for glycoprotein production. The selection of an appropriate production system depends on the intended applications and desired characteristics of the protein. Whether the goal is to produce glycoproteins mimicking native conditions or to intentionally alter glycan structures for specific purposes, such as enhancing immunogenicity in vaccines, understanding glycosylation present in the different systems and in different growth conditions is essential. This chapter will cover Escherichia coli, baculovirus/insect cell systems, Pichia pastoris, as well as different mammalian cell culture systems including Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) cell lines, and baby hamster kidney (BHK) cells.

Boosting sugarcane trash decomposition: synergistic action and proximity effect of xylanase and feruloyl esterase co-displayed on the cell surface of Pichia pastoris (Komagataella phaffi)

Xylanase and feruloyl esterase co-displayed on yeast surface boosts lignocellulosic biomass degradation through synergistic effect caused by their proximity.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein 9 in Pichia pastoris: The Combination of Gene Editing and Biological Manufacturing

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Protein 9 in Pichia pastoris: The Combination of Gene Editing and Biological Manufacturing

Functional analysis of a novel endo-β-1,6-glucanase MoGlu16 and its application in detecting cell wall β-1,6-glucan of Magnaporthe oryzae.

As an essential component of the fungal cell wall, β-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel β-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous β-1,6-glycosidic bonding polysaccharides. Based on this, β-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 105/ml conidia with 0.03 μg/μl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure.

Upregulation of TCPTP in Macrophages Is Involved in IL-35 Mediated Attenuation of Experimental Colitis.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease with complex etiology. Interleukin-35 (IL-35), as a cytokine with immunomodulatory function, has been shown to have therapeutic effects on UC, but its mechanism is not yet clear. Therefore, we constructed Pichia pastoris stably expressing IL-35 which enables the cytokines to reach the diseased mucosa, and explored whether upregulation of T-cell protein tyrosine phosphatase (TCPTP) in macrophages is involved in the mechanisms of IL-35-mediated attenuation of UC. After the successful construction of engineered bacteria expressing IL-35, a colitis model was successfully induced by giving BALB/c mice a solution containing 3% dextran sulfate sodium (DSS). Mice were treated with Pichia/IL-35, empty plasmid-transformed Pichia (Pichia/0), or PBS by gavage, respectively. The expression of TCPTP in macrophages (RAW264.7, BMDMs) and intestinal tissues after IL-35 treatment was detected. After administration of Pichia/IL-35, the mice showed significant improvement in weight loss, bloody stools, and shortened colon. Colon pathology also showed that the inflammatory condition of mice in the Pichia/IL-35 treatment group was alleviated. Notably, Pichia/IL-35 treatment not only increases local M2 macrophages but also decreases the expression of inflammatory cytokine IL-6 in the colon. With Pichia/IL-35 treatment, the proportion of M1 macrophages, Th17, and Th1 cells in mouse MLNs were markedly decreased, while Tregs were significantly increased. In vitro experiments, IL-35 significantly promoted the expression of TCPTP in macrophages stimulated with LPS. Similarly, the mice in the Pichia/IL-35 group also expressed more TCPTP than that of the untreated group and the Pichia/0 group.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

Cloning and characterization of bgl6111 gene encoding β‐glucosidase from bagasse metagenome

β‐Glucosidase (BGL) is an essential enzyme for the hydrolysis of cellulose in industrial processes, but natural BGL enzymes are poorly understood. Metagenomics is a robust tool for bioprospecting in the search for novel enzymes from the entire community’s genomic DNA present in nature. The metagenomics approach simplifies the process of searching for new BGL enzymes by extracting DNA and retrieving its gene information through a series of bioinformatic analyses. In this study, we report the gene cloning, heterologous expression of the bgl6111 gene (accession number MW221260) in Pichia pastoris KM71, and the biochemical characterization of the recombinant enzyme. We successfully identified the bgl6111 sequence of 2,520 bp and 839 amino acids with a molecular size of 89.4 kDa. The amino acid sequence of the bgl6111 gene showed 67.61% similarity to BGL from an uncultured bacterium (ABB51613.1). The BGL product has the highest activity on the third day at 1.210 U/mL, categorized as low production. The enzymatic activity could enhance up to 539.8% of 7.742 U/mL by using the ultrafiltration method. Our findings provide insightful information that bgl6111 obtained from bagasse metagenome could be an alternative candidate for industrial applications in the future.

Complete decomposition of poly(ethylene terephthalate) by crude PET hydrolytic enzyme produced in Pichia pastoris

Using enzymes to decompose poly(ethylene terephthalate) (PET) is an attractive strategy to the sustainable utilization of PET, and an effective production platform of PET degrading enzyme is a prerequisite to achieve this goal. Here, we exploited the industrial yeast strain Pichia pastoris to produce a potent PET hydrolase termed FAST-PETase, whose performance was further elevated by removing two N-linked glycosylations through molecular engineering. The expression of the yielded variant, FAST-PETase-212/277, was elevated by antibiotics selection and chaperon co-expression to exceed 3 g/L in a 30-L fermenter. Notably, the crude fermentation product can be directly applied to decompose PET without purification. More than 95 % postconsumer PET can be achieved by 0.5 mgenzyme gPET-1 in 24 h in a 10-L reaction system in a reactor. These results demonstrate the economic viability of producing PET hydrolytic enzyme with modern fermentation facilities for large scale PET decomposition.

Fine-Tuned Gene Expression Elements from Hybrid Promoter Libraries in Pichia pastoris.

As a desirable microbial cell factory, Pichia pastoris has garnered extensive utilization in metabolic engineering. Nevertheless, the lack of fine-tuned gene expression components has significantly constrained the potential scope of applications. Here, a gradient strength promoter library was constructed by random hybridization and high-throughput screening. The hybrid promoter, phy47, performed best with 2.93-fold higher GFP expression levels than GAP. The broad applicability of the novel hybrid promoter variants in biotechnological production was further validated in the biosynthesis of pinene and rHuPH20 with higher titers. The upstream regulatory sequences (UASE and URSD) were identified and applied to promoters GAP and ENO1, resulting in a 34 and 43% increase and an 18 and 37% decrease in the expression level, respectively. Yeast one-hybrid analysis showed that transcription factor HAP2 activates the hybrid promoter through a direct interaction with the crucial regulatory region UASH. Furthermore, a short segment of tunable activation sequence (20 bp) was also screened, and artificial promoters were constructed in tandem with the addition of regulatory sequence, resulting in a 61% expansion of the expression range. This study provides a molecular tool and regulatory elements for further synthetic biology research in P. pastoris.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

AbstractThe yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

The Pichia pastoris enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass

The demand for industrial enzymes is continually rising, fueled by the growing need to shift towards more sustainable industrial processes. However, making efficient enzyme production strains and identifying optimal enzyme expression conditions remains a challenge. Moreover, the production of the enzymes themselves comes with unavoidable impacts, e.g., the need to utilize secondary feedstocks. Here, we take a more holistic view of bioprocess development and report an integrative approach that allows us to rapidly identify improved enzyme expression and secretion conditions and make use of cyanobacterial waste biomass as feed for supporting Pichia pastoris fermentation. We demonstrate these capabilities by producing a phytase secreted by P. pastoris that is grown on cyanobacterium hydrolysate and buffered glycerol-complex (BMGY) medium, with genetic expression conditions identified by high-throughput screening of a randomized secretion library. When our best-performing strain is grown in a fed-batch fermentation on BMGY, we reach over 7 000 U/mL in three days.

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris

Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using PAOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.