Showing papers on "Pichia pastoris published in 1994"

Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.

Abstract: Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

Abstract: Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences.

Abstract: We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2·5 and 0·9 g/l respectively.

Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis.

Abstract: Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.

The positive and negative cis-acting elements for methanol regulation in the Pichia pastoris AOX2 gene

Abstract: The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2. The AOX2 gene is transcribed at a much lower level than the AOX1 gene. Apart from this difference in expression levels, the two genes are regulated similarly. To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives. By analyzing the expression of HSA in P. pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter. The positive cis-acting element AOX2-UAS, located between positions −337 and −313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter. A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast. Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter. The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.

The Pichia pastoris HIS4 gene: nucleotide sequence, creation of a non-reverting his4 deletion mutant, and development of HIS4-based replicating and integrating plasmids.

Abstract: We have obtained a clone of the Pichia pastoris HIS4 gene and have determined its nucleotide sequence. Based upon its deduced amino-acid sequence, the product of the P. pastoris HIS4 gene has the same structural organization as the Saccharomyces cerevisiae His4 protein and appears to encode a trifunctional enzyme catalyzing the second (phosphoribosyl-ATP pyrophosphohydrolase), third (phosphoribosyl-AMP cyclohydrolase), and tenth (histidinol dehydrogenase) steps in histidine biosynthesis. The chromosomal copy of the HIS4 gene was disrupted by homologous recombination, creating the strain SGY58. The his4Δ deletion mutation in this strain lacks the entire coding region of this gene and has a reversion rate that is undetectable. A set of complementary plasmids that carry the HIS4 gene was also developed. Among these are nine E. coli-P. pastoris shuttle vectors that transform the His4Δ deletion mutant at high efficiency and an integration vector for creating site-specific alterations of the P. pastoris genome.

Enzymatic Production of Glyoxal from Ethylene Glycol Using Alcohol Oxidase from Methanol Yeast.

Abstract: A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent Kms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent Km for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.

Importance of solute partitioning in biphasic oxidation of benzyl alcohol by free and immobilized whole cells of pichia pastoris.

Abstract: Using free and immobilized whole cells of Pichia pastoris, the biocatalytic oxidation of benzyl alcohol was investigated in different two-phase systems. This reaction was strongly influenced by both the substrate and product inhibitions, and the production rate of benzaldehyde in the aqueous system became maximum at the initial substrate concentration of ca. 29 g/L with the aldehyde formation less than 4 to 5 g/L even after a longer reaction period. The reaction rates in the two-liquid phase systems were predominantly determined by the partitioning behaviors of the substrate and product between the two phases rather than by enzyme deactivation by the organic solvents. In the two-liquid phase systems, consequently, the organic solvent acted as a reservior to reduce these inhibitory effects, and it was essential to select the organic solvent providing the optimal partitioning of the substrate into the aqueous phase as well as the preferential extraction of the product into the organic phase. The whole cells immobilized in a mixed matrix composed of silicone polymer [>50% (v/v)] and Ca alginate gel (<50%) worked well in the xylene and decane media, providing comparable activities with the free cells. The production rate of aldehyde was also influenced by the solute partitioning into the hydrophilic alginate phase where the cells existed. (c) 1994 John Wiley & Sons, Inc.

Glyoxylic acid/aminomethylphosphonic acid mixtures prepared using a microbial transformant

Abstract: An improved enzymatic process for reacting glycolic acid and oxygen in an aqueous solution in the presence of aminomethylphosphonic acid wherein the improvement comprises using a microbial cell catalyst that expresses glycolate oxidase (e.g., Pichia pastoris, Hansenula polymorpha, Aspergillus nidulans, or Escherichia coli) and endogenous catalase; soluble catalase may also be included. The resulting mixtures are useful intermediates in the production of N-(phosphonomethyl)glycine.

Pichia pastoris alcohol oxidase ZZA1 and ZZA2 regulatory regions for heterologous gene expression

Abstract: Two novel genomic clones, ZZA1 and ZZA2, were isolated which encode for Pichia pastoris alcohol oxidase isozymes. The 5' non-coding region of ZZA1 contains common structural features involved in the transcription and translation of eukaryotic genes. Comparison of the nucleotide sequences of the ZZA1 and AOX1 51 noncoding regions showed that they are 66% similar to each other. The rice α-amylase gene OS103 was placed under the transcriptional control of the ZZA1 promoter. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTGNNNGCTTCCAANNNNNTGGT) (SEQ ID NO:2) was found in the 51 flanking region. A yeast strain containing the ZZA1 -OS103 fusion and secreting biologically active a-amylase into the culture media while converting starch to ethanol was produced. The ZZA1 and ZZA2 regulatory sequences may be used to contol the expression of other heterologous proteins in multiple yeast species. Methods of purifying proteins that are regulators of alcohol oxidase expression (referred to as AOER proteins) and methods of isolating these proteins are also provided. The invention also provides for the efficient conversion of starch into ethanol.

Dextranase enzyme, method for its production and DNA encoding the enzyme

Abstract: A method for the isolation and expression of a gene coding for dextranase of the fungus Penicillium minioluteum. This enzyme of fungal origin is produced by expression at high levels in yeast. For this purpose, a cDNA copy of the mRNA coding for dextranase enzyme of the Penicillium minioluteum fungus was isolated and sequenced. This cDNA was transfered into Pichia pastoris cells. Recombinant yeasts capable of secreting dextranase to the culture medium were obtained thereby. The dextranase enzyme obtained can be used, e.g. in the sugar industry to hydrolyze the dextran in cane juices to increase the sugar production.

PICHIA PASTORIS ALCOHOL OXIDASE ZZA1 and $i(ZZA2)

Abstract: Two novel genomic clones, ZZA1 and ZZA2, were isolated which encode for Pichia pastoris alcohol oxidase isozymes. The 5' non-coding region of ZZA1 contains common structural features involved in the transcription and translation of eukaryotic genes. Comparison of the nucleotide sequences of the ZZA1 and AOX1 51 noncoding regions showed that they are 66 % similar to each other. The rice α-amylase gene OS103 was placed under the transcriptional control of the ZZA1 promoter. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTGNNNGCTTCCAANNNNNTGGT) (SEQ ID NO:2) was found in the 51 flanking region. A yeast strain containing the ZZA1-OS103 fusion and secreting biologically active a-amylase into the culture media while converting starch to ethanol was produced. The ZZA1 and ZZA2 regulatory sequences may be used to control the expression of other heterologous proteins in multiple yeast species. Methods of purifying proteins that are regulators of alcohol oxidase expression (referred to as AOER proteins) and methods of isolating these proteins are also provided. The invention also provides for the efficient conversion of starch into ethanol.

Mutant strain of Pichia pastoris which utilizes methanol in the presence of glucose

Abstract: A methylotrophic and glucotrophic mutant strain capable of producing a heterologous protein and a method for producing a heterologous protein, comprising culture of the mutant strain. The mutant strain of the present invention can be grown in a medium containing both methanol and glucose, with the effect that the growth of the strain and production of a heterologous protein proceed at the same time. Accordingly, a heterologous protein can be produced in a large amount in a short time.

Expression of beta-2-microglobulin in pichia pastoris

Abstract: High levels of recombinant beta-2 microglobulin can be expressed from methylotropic yeasts, particularly Pichia pastoris.

Production of bacillus insect poison in yeast methylotroph

Abstract: A method for producing one or more Bacillus toxin polypeptides by culturing methylotrophic yeast cells which have a gene(s) capable of expressing the Bacillus toxin polypeptide(s) in such cells under conditions that the gene(s) is/are transcribed is provided. The toxin polypeptide encoding segment of the gene(s) has a G+C content of about 40%-55%, and preferably comprises methylotrophic yeast codons. The preferred species of yeast for expressing such synthetic Bacillus toxin gene(s) is Pichia pastoris. Bacillus toxin polypeptides encoded by synthetic genes are expressed at high levels in transformed methylotrophic yeast cell. The toxin expressing cells may be administered as live cells or heat-killed whole cells to provide an insecticidal composition for killing susceptible insect larvae. Also provided by the present invention are DNAs capable of transforming methylotrophic yeast to express one or more Bacillus toxin polypeptides, cultures of such yeast cells transformed with such DNAs and novel Bacillus toxin polypeptides made by the method of the invention. The transformed yeast cells of the present invention are readily ingested as food by insect larvae which are susceptible to the toxin polypeptides.

EXPRESSION DE BETA-2-MICROGLOBULINE DANS $i(PICHIA PASTORIS)

Abstract: Des taux eleves de beta-2-microglobuline de recombinaison peuvent etre exprimes a partir de levures methylotropiques, notamment Pichia pastoris.