Pichia Pastoris Strain Research Articles

Low cost production of perdeuterated biomass using methylotrophic yeasts

AbstractThree strains of methylotrophic yeasts, Candida boidinii, Pichia angusta (previously Hansenula polymorpha) and Pichia Pastoris, were studied for their capacity to grow on methanol in deuterated media. Growth rates, determined relative to the extent of deuteration of water and/or methanol, showed that water deuteration was the major limiting factor. After adaptation to deuterium by progressive transfer through media of increasing deuteration, growth rates were diminished relative to those obtained on hydrogenated media of identical salts composition: the two Pichia species retained the highest growth rates (μ=0.02 h−1) in the fully deuterated medium. Perdeuterated biomass (16 g) was obtained in a 1 liter fed‐batch fermentation and the extent of deuteraton of isolated ergosterol has been shown to be 97.5% by mass spectrometric analysis.

Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies

We have constructed a synthetic secretion cassette encoding the α-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived α-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1 % Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 μg/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797–802]. In addition, the full-length protein, mEGF 1-53, was secreted by P. pastoris.

High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.

We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.

High-level secretion of biologically active aprotinin from the yeast Pichia pastoris.

A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Invertase gene ( SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc −) and that introduction of the invertase gene ( SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc +. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc + transformants. It was shown that the Suc + transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc − segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc + cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc + phenotype was also observed. However, stable Suc + transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc + phenotype after 20 generations of growth in a nonselective medium.

Isolation and characterization of a methanol utilizing yeast with high cell yield

A yeast (Pichia pastoris strain CMB 10) growing on methanol as sole carbon and energy source was isolated. The high growth rate (0.235·h−1 at pH 5) and high cell yield (0.41 g cell per g methanol at pH 3.5) of this strain are of interest for production of single cell protein (SCP). Other advantages of the strain are: low maintenance coefficient (m=9.5 mg·g−1·h−1), high affinity for methanol (Ks=100 mg·l−1), possibility of non aseptic culture at low pH (pH 3.5), equilibrated amino acid profile and flocculating properties.

DETERMINATION OF METHANOL STRONGLY ASSIMILATING YEASTS

Hundreds of strains of methanol-assimilating yeasts were isolated in pure culture from a wide variety of natural sources through investigations on industrial cell production and the metabolism of methanol. Seven of these strains showed heavy growth on a methanol medium. Taxonomic studies on these strongly methanol-assimilating yeasts indicated that five strains were the same species and belonged to a new species, Pichia methanolica, one strain Pichia pastoris, and another Candida boidinii.