Showing papers on "Pichia pastoris published in 2007"

Expression of recombinant proteins in Pichia pastoris.

Abstract: Pichia pastoris has been used extensively and successfully to express recombinant proteins. In this review, we summarize the elements required for expressing heterologous proteins, and discuss various factors in applying this system for protein expression. These elements include vectors, host strains, heterologous gene integration into the genome, secretion factors, and the glycosylation profile. In particular, we discuss and evaluate the recent progress in optimizing the fermentation process to improve the yield and stability of expressed proteins. Optimization can be achieved by controlling the medium composition, pH, temperature, and dissolved oxygen, as well as by methanol induction and feed mode.

Transcriptomics-Based Identification of Novel Factors Enhancing Heterologous Protein Secretion in Yeasts

Abstract: Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.

Abstract: Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris.

Abstract: In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l−1 in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.

Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions

Abstract: It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.

Cloning and functional expression of thermostable beta-glucosidase gene from Thermoascus aurantiacus.

Abstract: A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.

Tilapia follicle-stimulating hormone (FSH): immunochemistry, stimulation by gonadotropin-releasing hormone, and effect of biologically active recombinant FSH on steroid secretion.

Abstract: In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17–18 kDa and 26–30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanc...

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates

Abstract: The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39×10−5 s−1 and 4.44×10−5 s−1) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

Norovirus capsid protein expressed in yeast forms virus-like particles and stimulates systemic and mucosal immunity in mice following an oral administration of raw yeast extracts.

Abstract: Norovirus (NV) gastroenteritis is a widespread disease affecting people of all ages worldwide. A simple, safe, and easily deliverable vaccine may be the key for the control and prevention of NV gastroenteritis. In this study, we demonstrated that a NV recombinant capsid protein (strain VA387, genogroup II.4) expressed in yeast (Pichia pastoris) spontaneously formed virus-like particles (VLPs) like those expressed in other in vitro systems. Oral administration of raw material from the yeast cell lysates containing 0.1 mg of VLPs without an adjuvant resulted in systemic and mucosal immune responses in mice. Significantly higher and earlier responses were observed in mice receiving a higher dose (1 mg per dose) of the antigen. Both the serum and fecal antibodies blocked VA387 VLP binding to its histo-blood group antigen receptors. The animals did not reveal any side effect following the administration of the yeast lysates. Our results indicated that yeast is a simple, effective alternative for NV VLP production. The mice immunization study also indicated that the oral administration of raw yeast extracts without an adjuvant is a safe and simple way which is worth to be studied for vaccine delivery in humans.

Preparation of an Amino Acid Intermediate for the Dipeptidyl Peptidase IV Inhibitor, Saxagliptin, using a Modified Phenylalanine Dehydrogenase

Abstract: The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase, and further scaled up using a preparation of the two enzymes expressed in E. coli.

Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina.

Abstract: A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25 degrees C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml(-1). The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40 degrees C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The Km and Vmax values for birchwood xylan are 2.06 mg ml(-1) and 0.49 mmol min(-1)mg(-1), respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.

Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris

Abstract: Beta-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant beta-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding beta-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, beta-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l(-1). The beta-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized beta-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45 degrees C, respectively.

Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

Abstract: Background: The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL. Results: The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected. Conclusion: The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-ofprinciple, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.

Translation elongation factor 1-α gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter

Abstract: The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (PTEF1) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) by using a bacterial lipase gene as a reporter gene. PTEF1 demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than PGAP. The sequence of the gene was deposited in GenBank under accession no. EF014948.

The proteolytic systems and heterologous proteins degradation in the methylotrophic yeastPichia pastoris

Abstract: ThePichia pastoris expression system has been successfully used for production of various recombinant heterogeneous proteins. The productivity ofP. pastoris can be improved substantially by bioreactor cultivations. However, heterologous proteins degradation increases as well in high-cell density culture. Proteolytic degradation is a serious problem since the yeast has been employed to express recombinant proteins. In this review, some of the recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels on the cytosolic proteasome, vacuolar proteases, and proteases located within the secretory pathway inP. pastoris, are reviewed.

Gene cloning, overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05

Abstract: Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.