Protein In Pichia Pastoris Research Articles

Optimizing Pichia pastoris protein secretion: Role of N-linked glycosylation on the α-mating factor secretion signal leader

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

Identification of a novel growth-associated promoter for biphasic expression of heterogenous proteins in Pichia pastoris.

Pichia pastoris (P. pastoris) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (PAOX1) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for PAOX1, methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (PFDH800). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the PFDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and PAOX1, respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by PFDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of PAOX1. Our PFDH800-based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the PAOX1 promoter.IMPORTANCEThe alcohol oxidase 1 promoter (PAOX1) expression system has the characteristics of biphasic expression and high expression levels, making it the most widely used promoter in the yeast Pichia pastoris. However, PAOX1 requires methanol induction, which can be toxic and poses a fire hazard in large quantities. Our research has found that the activity of PFDH800 is closely related to the growth state of cells and can achieve biphasic expression without the need for an inducer. Compared to other reported non-methanol-induced biphasic expression systems, the system based on the PFDH800 offers several advantages, including high expression levels, simple construction, minimal impact on cellular metabolism, no need for an inducer, and the ability to fine-tune expression.

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.

AOX1 promoter-driven expression of yeast-enhanced green fluorescent protein in Pichia pastoris

Green fluorescent protein (GFP) is a widely employed reporter for studying various cellular functions in genetic engineering. Methylotrophic yeast; Pichia pastoris is an excellent host for heterologous protein production. Codon optimized yeast-enhanced green fluorescent protein (yEGFP) has been reported to have higher expression as a yeast reporter which can be used to study many aspects of P. pastoris. The present investigation was focused on studying the yEGFP intracellular expression pattern in the P. pastoris GS115 strain. yEGFP is cloned into the pPICZ A vector under the control of an AOX1 (Alcohol Oxidase) promoter that was induced by adding methanol to the growth medium. However, methanol concentration and time of induction could affect the optimum expression of yEGFP. Hence, those conditions were tested for manifesting optimum yEGFP expression by monitoring the fluorescence intensities in minimal methanol histidine media supplemented with 0.5%, 1%, 2%, and 2.5% methanol. The pH of the media and the composition of the media (minimal or complex) also affect recombinant protein production. The effect of pH and the media composition was investigated in buffered media at pH 6 and unbuffered media of minimal methanol histidine media and methanol complex media. The results showed that the yEGFP expression increased up to the 5th day of induction and then decreased on the 6th day, 2% methanol showed the highest expression, and the pH and the media composition did not have any effect on the yEGFP expression. In conclusion, the optimum yEGFP expression can be obtained in 2% methanol on the 5th day of induction irrespective of minimal or complex media used.

Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris.

The loss of mixing efficiency inherent to bioreactor process operated at large-scale yields to the formation of concentration gradient and thus to heterogeneous culture conditions. For processes operated with methanol feeding, P. pastoris faces oscillatory culture conditions that significantly affect the cell ability to produce secretory recombinant proteins at high yield. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found in the upper part of the bioreactor near the feeding point, triggers the unfolded protein response (UPR) and thus impairs proper protein secretion. Methanol co-feeding with sorbitol was shown herein to reduce the UPR response and to restore productivity of secreted protein.

Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris.

The heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production usually requires to express more than one gene in the host cells. In eukaryotes, the pathway flux is typically balanced by controlling the transcript levels of the genes involved. It is difficult to balance the stoichiometric fine-tuning of the reaction steps of the pathway by acting on one or two promoters. Furthermore, the promoter used should not be identical to avoid loss of inserted genes by recombination or dilute its transcription factors. Based on RNA-seq data, 18 candidate genes with the highest transcription levels at three carbon sources (glucose, glycerol and methanol) were selected and their promoter regions were isolated from GS115 genome. The performance of these promoters on the level of protein production was evaluated using LacZ and EGFP genes as the reporters, respectively. These isolated promoters all exhibited activity to express LacZ gene. Using LacZ as a reporter, of the 18 promoter candidates, 9 promoters showed higher expression levels for the reporter compare to pGAP, a strong promoter widely used for constitutive expression of heterologous proteins in Pichia pastoris. These promoters with high expression levels were further employed to evaluate secreted expression using EGFP as a reporter. 6 promoters exhibited stronger protein expression compare to pGAP. Interestingly, the protein expression driven by pFDH1 was slightly higher than that of commonly used pAOX1 at methanol, and methanol-induced expression of pFDH1 was not repressed by glycerol. The various promoters identified in this study could be used for heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production. the methanol-induced pFDH1 that is not repressed by glycerol is an attractive alternative to pAOX1 and may provide a novel way to produce heterologous proteins in Pichia pastoris.

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

Challenges of expressing recombinant human tissue factor as a secreted protein in Pichia pastoris

Tissue factor (TF) is the core reagent in the prothrombin time (PT) assay. In this study, expression and α-factor mediated secretion of three forms of tissue factor (full-length TF (Full-TF), extracellular plus transmembrane domain (TED-TF), and only extracellular domain (ED-TF) were investigated in Pichia pastoris. The amino acid sequence of TF was obtained from the UniProt database, back-translated and codon-optimized for expression in Pichia pastoris. The Full-TF sequence was synthesized but the ED-TF, TED-TF coding fragments were extracted from the Full-TF by PCR. All the coding sequences were cloned into pPICZαA vector in-frame with the α-factor; and electroporated into KM71H. The culture supernatants and the cell lysates were analyzed using SDS-PAGE, dot-blotting, and Western-blotting for expression of TF. The Full-TF and TED-TF expression vector pPICZαA were successfully inserted into the KM71H, but the product was not detected in the SDS-PAGE analysis of the culture supernatant. However, ED-TF expression and secretion was verified by SDS-PAGE, dot blotting, and Western blotting. It seems that the TM domain in the Full-TF and TED-TF have an important role in impairing α-factor-mediated secretion of TF. Therefore, further investigation is necessary to overcome challenges of expressing Full-TF as a heterologous protein in P. pastoris.

Going beyond the limit: Increasing global translation activity leads to increased productivity of recombinant secreted proteins in Pichia pastoris

Yeasts are widely used cell factories for commercial heterologous protein production, however, specific productivities are usually tightly coupled to biomass formation. This greatly impacts production processes, which are commonly not run at the maximum growth rate, thereby resulting in suboptimal productivities. To tackle this issue, we evaluated transcriptomics datasets of the yeast Pichia pastoris (syn. Komagataella phaffii), which is known for its high secretory efficiency and biomass yield. These showed a clear downregulation of genes related to protein translation with decreasing growth rates, thus revealing the yeast translation machinery as cellular engineering target. By overexpressing selected differentially expressed translation factors, translation initiation was identified to be the main rate-limiting step. Specifically, overexpression of factors associated with the closed-loop conformation, a structure that increases stability and rates of translation initiation before start codon scanning is initiated, showed the strongest effects. Overexpression of closed-loop factors alone or in combination increased titers of different heterologous proteins by up to 3-fold in fed-batch processes. Furthermore, translation activity, correlating to the obtained secreted recombinant protein yields, selected transcript levels and total protein content were higher in the engineered cells. Hence, translation factor overexpression, globally affects the cell. Together with the observed impact on the transcriptome and total protein content, our results indicate that the capacity of P. pastoris for protein production is not at its limit yet.

Production and Preparation of Isotopically Labeled Human Membrane Proteins in Pichia pastoris for Fast-MAS-NMR Analyses.

Membrane proteins (MPs) comprise about one-third of the human proteome, playing critical roles in many physiological processes and associated disorders. Consistently, they represent one of the largest classes of targets for the pharmaceutical industry. Their study at the molecular level is however particularly challenging, resulting in a severe lack of structural and dynamic information that is hindering their detailed functional characterization and the identification of novel potent drug candidates.Magic Angle Spinning (MAS) NMR is a reliable and efficient method for the determination of protein structures and dynamics and for the identification of ligand binding sites and equilibria. MAS-NMR is particularly well suited for MPs since they can be directly analysed in a native-like lipid bilayer environment but used to require aggravating large amounts of isotope enriched material. The frequent toxicity of human MP overexpression in bacterial cultures poses an additional hurdle, resulting in the need for alternative (and often more costly) expression systems. The recent development of very fast (up to 150kHz) MAS probes has revolutionized the field of biomolecular solid-state NMR enabling higher spectral resolution with significant reduction of the required sample, rendering eukaryotic expression systems cost-effective.Here is presented a set of accessible procedures validated for the production and preparation of eukaryotic MPs for Fast-MAS 1H-detected NMR analysis. The methodology is illustrated with the human copper uptake protein hCTR1 recombinantly produced and 13C-15N uniformly labeled with the versatile and affordable Pichia pastoris system. Subsequent purification procedures allow the recovery of mg amounts that are then reconstituted into liposome formulations compatible with solid-state NMR handling and analysis.

Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System.

Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL−1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

BackgroundCodon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias.ResultsIn this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris.ConclusionsCodon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

Half-life extension of efficiently produced DARPin serum albumin fusions as a function of FcRn affinity and recycling

Serum albumin shows slow clearance from circulation due to neonatal Fc receptor (FcRn)-mediated recycling and has been used for half-life extension. We report here fusions to a high-affinity DARPin, binding to Epithelial Cell Adhesion Molecule (EpCAM). We developed a novel, efficient expression system for such fusion proteins in Pichia pastoris with titers above 300 mg/L of lab-scale shake-flask culture. Since human serum albumin (HSA) does not bind to the murine FcRn, half-lives of therapeutic candidates are frequently measured in human FcRn transgenic mice, limiting useable tumor models. Additionally, serum albumins with extended half-life have been designed. We tested HSA7, motivated by its previously claimed extraordinarily long half-life in mice, which we could not confirm. Instead, we determined a half-life of only 29 h for HSA7, comparable to MSA. The fusion of HSA7 to a DARPin showed a similar half-life. To rationalize these findings, we measured binding kinetics and affinities to murine and human FcRn. Briefly, HSA7 showed affinity to murine FcRn only in the micromolar range, comparable to MSA to its cognate murine FcRn, and an affinity in the nanomolar range only to the human FcRn. This explains the comparable half-life of MSA and HSA7 in mice, while wild-type-HSA has a half-life of only 21 h, as it does not bind the murine FcRn and is not recycled. Thus, HSA-fusions with improved FcRn-affinity, such as HSA7, can be used for preclinical experiments in mice when FcRn transgenes cannot be used, as they reflect better the complex FcRn-mediated recycling and distribution mechanisms.

Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris.

Background:The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Los1 (loss of supression) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends RLS. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods:A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 ScFv expression were compared between the parent and mutant strains.Results:The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively.Conclusion:The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.

Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300 was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes.

Selection and in vitro and in vivo characterization of a Kunitz protease inhibitor domain of protease nexin 2 variant that inhibits factor XIa without inhibiting plasmin

The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.

Expression of proteins in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial enzymes. The popularity of P. pastoris stems from its ability to grow to high cell densities, producing high titers of secreted heterologous protein with very low amounts of endogenous proteins. Its ability to express correctly folded proteins with post-translational modifications makes it an excellent candidate for the production of biopharmaceuticals. In addition, production in P. pastoris typically uses the strong, methanol-inducible and tightly regulated promoter (PAOX1), which can result in heterologous protein that constitutes up to 30% of total cell protein upon growth in methanol. In this chapter, we present methodology for the production of secreted recombinant proteins in P. pastoris, and we discuss alternatives to enhance protein production with the desired yield and quality.

PsAA9A, a C1-specific AA9 lytic polysaccharide monooxygenase from the white-rot basidiomycete Pycnoporus sanguineus.

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

Construction and screening of a glycosylphosphatidylinositol protein deletion library in Pichia pastoris

BackgroundGlycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches.ResultsThrough an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the ‘compensatory mechanism’, which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity.ConclusionsThis GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions.