scispace - formally typeset
Search or ask a question
Topic

Pichia pastoris

About: Pichia pastoris is a research topic. Over the lifetime, 7937 publications have been published within this topic receiving 162645 citations. The topic is also known as: Komagataella pastoris.


Papers
More filters
Journal ArticleDOI
TL;DR: The isolation and functional analysis of the gene encoding asparagus 6G-FFT was reported, and the deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants.
Abstract: * Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.

53 citations

Journal ArticleDOI
TL;DR: This study lays a solid foundation for the commercial preparation of 10-deacetyltaxol through the recombinant yeast and provides a successful paradigm for scaling-up HCDF of P. pastoris to the demonstration scale.
Abstract: Scaling-up of high-cell-density fermentation (HCDF) of Pichia pastoris from the lab or pilot scale to the demonstration scale possesses great significance because the latter is the final technological hurdle in the decision to go commercial. However, related investigations have rarely been reported. In this paper, we study the scaling-up processes of a recombinant P. pastoris from the pilot (10 to 100-L) to the demonstration (1,000-L) scales, which can be used to convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol by the β-xylosidase for semi-synthesis of Taxol. We demonstrated that a pure oxygen supplement can be omitted from the HCDF if the super atmospheric pressure was increased from 0.05 to 0.10 ± 0.05 MPa, and we developed a new methanol feeding biomass-stat strategy (0.035 mL/g/h) with 1% dissolved oxygen and 100 g/L initial induction biomass (dry cell weight). The scaling-up was reproducible, and the best results were obtained from the 1,000-L scale, featuring a shorter induction time and the highest enzyme activities and productions, respectively. The specific growth and specific production rates were also determined. This study lays a solid foundation for the commercial preparation of 10-deacetyltaxol through the recombinant yeast. It also provides a successful paradigm for scaling-up HCDF of P. pastoris to the demonstration scale.

53 citations

Journal ArticleDOI
TL;DR: The results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.
Abstract: The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an A. oryzae library by using the A. fumigatus dppIV gene as a probe. Reverse transcriptase PCR experiments showed that the A. oryzae dppIV gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.

53 citations

Journal ArticleDOI
TL;DR: It is suggested that the stability of the protein arising from the hydrophobic effect is reduced by the C121S mutation so that unfolded or partially unfolded states are more favored.
Abstract: The lipocalin β-lactoglobulin (BLG) is the major whey protein of bovine milk and is homodimeric at physiological conditions. Each monomer contains two disulfide bonds and one cysteine at position 121 (C121). This free thiol plays an important role in the heat-induced aggregation of BLG and, possibly, in its conformational stability. We describe here the expression in the yeast Pichia pastoris of a mutant bovine BLG, in which C121 was changed into Ser (C121S). Circular dichroism and high-performance liquid chromatography experiments, together with the X-ray crystal structure, show that the C121S mutant retains a nativelike fold at both neutral and acid pH. The mutation completely blocks the irreversible aggregation induced by heat treatment at 90 °C. Compared to the recombinant wild-type protein, the mutant is less stable to temperature and disulfide reducing agents and is much more sensitive to peptic digestion. Moreover, its affinity for 1-anilino-8-naphthalenesulfonate is increased at neutral and acid p...

53 citations

Journal ArticleDOI
TL;DR: Batch fermentations were used to study the effect of different glycerol concentrations and pH conditions on growth of recombinantPichia pastoris, and there were no differences between Mut+ and Mut- strains during cell growth on Glycerol.
Abstract: Batch fermentations were used to study the effect of different glycerol concentrations and pH conditions on growth of recombinantPichia pastoris. Two strains ofP. pastoris were used: a wild-type in methanol utilization (Mut+) and a mutant defective in methanol utilization (Mut-). Under constant pH conditions of 5.0, glycerol concentrations up to 12% were efficiently utilized. Cell yield (Yx/s) of about 0.8 and a final cell density of about 95 g/L (dry cell) were achieved. However, there were significant differences (probability [Pr]> F 0.0351) in specific growth rates between the initial glycerol concentrations of 2, 7, and 12%. When fermentations were conducted without pH control, growth continued until the pH had decreased to about 2.5. Growth stopped at pH 2.2 with uncontrolled pH, and residual glycerol concentrations were greater than 2%. As a result, Yx/s decreased to about 0.3. There were no differences between Mut+ and Mut- strains during cell growth on glycerol.

53 citations


Network Information
Related Topics (5)
Plasmid
44.3K papers, 1.9M citations
85% related
Saccharomyces cerevisiae
32.1K papers, 1.6M citations
83% related
Escherichia coli
59K papers, 2M citations
83% related
Peptide sequence
84.1K papers, 4.3M citations
82% related
Complementary DNA
55.3K papers, 2.7M citations
81% related
Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023150
2022340
2021255
2020303
2019374
2018401