Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production

A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20 g/L glycerol, 7.5 g/L (NH4)2SO4, 1 g/L MgSO4.7H2O, 8.5 g/L KH2PO4, 1.5 mL/L vitamin solution and 20 mL/L trace metal solution. Using the optimized medium 11.25 g DCW/L biomass was produced giving a yield coefficient of 0.55 g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23 h(-1) and Monod saturation constant of 0.178 g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers.     

Modeling of mini-proinsulin production in Pichia pastoris using the AOX promoter

An unstructured model based on mass balance equations for a recombinant methylotrophic yeast Pichia pastoris Mut^S (Methanol Utilization Slow) strain expressing the mini-proinsulin (MPI), was successfully established in quasi-steady state fed-batch fermentations with varying total quantity of biomass in a 7 l fermenter. The model describes the relationships between the total biomass and induction time, both in the batch and fed-batch phases. In addition, good correlations were obtained when the total quantity of MPI was correlated with the total biomass, demonstrating that the product of interest is associated with growth in the methanol phase. The parameters of the fermentation model obtained are similar to those reported by other researchers.     

Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption

Endostatin, a 20 KDa fragment of collagen XVIII, was shown to have an inhibitory effect on angiogenesis and can potentially be used as a tumor growth suppressor. To obtain the amount needed for testing, the protein was successfully cloned and expressed in Pichia pastoris. At the end of the fermentation process, the concentration of the endostatin in the culture was 50 mg per liter, accompanied by 400 gr per liter (wet weight) of biomass. Before the protein can be captured and purified on a packed bed of heparin-Sepharose, the biomass must be removed. Because of the high biomass concentration, conventional biomass removal techniques like centrifugation or filtration are inefficient and cumbersome. Therefore, the expanded-bed adsorption technique was chosen as an alternative approach. An efficient procedure for the initial recovery and purification of the endostatin was developed. The process utilized a cation- exchanger resin instead of a heparin-based affinity resin, because its dynamic capacity was higher, even though it was affected by the high linear flow on the expanded bed. After adjusting the conductivity, pH and biomass concentration, the complete broth was pumped directly on the expanded-bed matrix (Streamline SP XL). Though the yields of protein are similar, the expanded-bed approach is superior to the packed-bed method for several reasons. The expanded-bed process was shorter (only 8 hours compared to 16 hours for the packed bed), it is cheaper, and the product has higher specific activity (29% compared with 18%). Endostatin produced by the expanded-bed adsorption method showed the expected bioactivity and is currently being tested for its potential as a tumor suppressor.     

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.     

Permeabilization of Pichia pastoris for glycolate oxidase activity

Maximum activity of glycolate oxidase was obtained from a recombinant Pichia pastoris by permeabilization with 0.1% benzalkonium chloride for 60 min at room temperature. After treatment, intracellular glycolate oxidase activity increased 10-fold with respect to untreated cells.     

A simple structured model for recombinant IDShr protein production in Pichia pastoris

A simple structured model is proposed for the methanol production phase of the iduronate 2-sulphate sulfatase recombinant enzyme (IDShr) in Pichia patoris Mut(+). The model is mainly focused in oxidative stress phenomenon due to methanol consumption and based on extracellular experimental information and the basic knowledge of methanol metabolism in Pichia pastoris yeast (P. pastoris). The model's prediction shows a reasonable accuracy as compared with the experimental data. Likewise, it was proved that this model is able to simulate the production of other recombinant protein in P. pastoris.     

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.     

Heterologous production of Aspergillus fumigatus keratinase in Pichia pastoris

Aspergillus fumigatus Fresenius was previously shown to grow in mineral medium containing chicken feather flour as carbon and nitrogen source. Substantial proteolytic keratin-degrading activity was present in the culture supernatant after 24–72 h of growth at 42 °C. The keratinase was successfully purified by a single ion exchange chromatographic procedure and had a molecular mass of 31 kDa as determined by SDS–PAGE. The keratinase cDNA was expressed in Pichia pastoris cells and the recombinant clones were shown to be able to produce substantial caseinolytic, azo-keratinolytic and keratinolytic activities. SDS–PAGE and Western-blotting analysis using antibody against keratinase of A. fumigatus showed the presence of a single protein in the culture supernatants of several recombinant P. pastoris cells. This protein had a molecular mass corresponding to that of the A. fumigatus keratinase. The enzyme production profile showed that theP. pastoris recombinant cells produced an increasing amount of proteolytic and azo-keratinolytic activities over a 72 h growth period. Dry weight determination analysis indicated that 10% of the keratin flour was hydrolysed over a 24 h incubation period with 510 U (caseinolytic activity) of the recombinant keratinase.     

Functional expression and production of human H-ferritin in Pichia pastoris

Human heavy chain ferritin (H-ferritin) was cloned from human heart cDNA library and expressed in Pichia pastoris. The H-ferritin transformant was cultivated by fed-batch and the cell mass reached about 52 g cell dry wt l^–1 after 150 h. In atomic absorption spectrometry analysis, intracellular content of iron in H-ferritin transformant was measured to 3038 ± 72 g g^–1 which was 9.6-fold more than that of control strain.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.     

Efficient secretion of human endostatin in the yeast, Pichia pastoris

Endostatin is a 20 kDa COOH-terminal fragment of collagen XVIII that inhibits angiogenesis and tumor growth. The cDNA coding for human endostatin in human fetal liver has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of human endostatin has been achieved (about 200 mg of endostatin in 1 l of culture). The recombinant human endostatin was purified to homogeneity by heparin-affinity column, and showed antiproliferative effect on rat brain micro-vascular endothelia cells.     

Increasing the cell viability and heterologous protein expression of Pichia pastoris mutant deficient in PMR1 gene by culture condition optimization

In this study, we assessed the potential of PMR1-disrupted Pichia pastoris (Pppmr1) expressing human serum albumin and interferon alpha2b fusion protein (HSA-IFN-alpha2b) in large-scale fermentation. The high osmotic pressure of standard basal salts medium (BSM) was detrimental to the growth and viability of Pppmr1. HSA-IFN-alpha2b was secreted into a supernatant with a concentration of up to 112 mg/L after 20 h of induction and then began to decline. In vitro stability tests indicated that the disappearance of HSA-IFN-alpha2b was ascribed to proteolytic degradation. Decreasing the salt concentration of BSM medium to one quarter of the original formula improved the growth and viability of Pppmr1. As a result of reduced cell lysis and protease release, HSA-IFN-alpha2b was stable in the supernatant, which enabled a longer production phase (30 h) and a higher expression level (215 mg/L). Lowering the culture temperature to 20°C increased the cell viability during carbon source transition and alleviated the oxygen and methanol limitation, which extended the production phase to 40 h and increased the expression level to 680 mg/L. The addition of 2% Soytone prolonged the production phase to 60 h and increased the expression level to 1,260 mg/L, which was more than tenfold higher than that of Pppmr1 cultured under the conditions recommended by Invitrogen.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Monitoring a recombinant Pichia pastoris fed batch process using Fourier transform mid-infrared spectroscopy (FT-MIRS)

The use of Fourier transform mid-infrared spectroscopy (FT-MIRS) to predict the concentrations of key analytes in fed-batch cultivations of an industrial strain of Pichia pastoris in a chemical complex medium was investigated. Models for glycerol, methanol (substrates), and product (an heterologous protein) were built, and evaluated. The use of a multi-bounce attenuated total reflectance (HATR) accessory aided spectral acquisition in optically dense samples. Generally, all models were robust and performed well on external validation, using data from processes not present in the original modelling exercise. Substrate models lacked the complexity of some previous IR models, and the models performed adequately even at low analyte concentration (<1 g l^–1). Thus, simultaneous, rapid monitoring of low concentrations of multiple analytes in a complex bioprocess matrix with little or no sample pre-treatment is achievable using ATR FT-MIRS.     

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Elevated expression of human alpha-1 antitrypsin mediated by yeast intron in Pichia pastoris

Intron-mediated enhancement has been documented in many cases to involve large positive effect on gene expression. To address this, human Alpha-1 antitrypsin (hAAT) gene was integrated into Pichia pastoris with and without a yeast intron generated from the final plasmid pBlu-exII-int-exIII and ligated into the EcoRI/BamHI multiple cloning site of the yeast shuttle vector pHIL-S1. The chimeric exon–intron complex in the middle of the naturally occurring hAAT exons II and III caused a 23-fold enhancement of hAAT expression in P. pastoris, measured through SDS-PAGE and immunoblot analyses.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.