Showing papers on "Pichia pastoris published in 1993"
TL;DR: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest and improvements in understanding and application have improved its utility even further.
Abstract: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.
993 citations
TL;DR: A 69%-synthetic gene with increased G + C content is designed and constructed which overcomes this transcriptional problem, giving rise to full-length mRNA, which indicates differences in mRNA 3'-end formation between the two yeasts.
183 citations
Journal Article•
TL;DR: Peroxisome loss during glucose repression is suggested to be due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.
116 citations
TL;DR: The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM- sensitive factor (NSF), and other related ATPases, and weak homology to a yeast myosin was also observed.
Abstract: The biogenesis and maintenance of cellular organelles is of fundamental importance in all eukaryotic cells. One such organelle is the peroxisome. The establishment of a genetic system to study peroxisome biogenesis in the methylotrophic yeast Pichia pastoris has yielded many different complementation groups of peroxisomal assembly (pas) or peroxisome-deficient (per) mutants. Each appears to be deficient in functional peroxisomes. One of these mutants, pas5, has been characterized, complemented, and the gene sequenced. Ultrastructural studies show that normal peroxisomes are not present in pas5, but aberrant peroxisomal structures resembling "membranous ghosts" are frequently observed. The "peroxisome ghosts" appear to be induced and segregated to daughter cells normally. Biochemical fractionation analysis of organelles of the pas5 mutant reveals that peroxisomal matrix enzymes are induced normally but are found mostly in the cytosol. However, purification of peroxisome ghosts from the mutant shows that small amounts (< 5%) of matrix enzymes are imported. The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM-sensitive factor (NSF), and other related ATPases. Weak homology to a yeast myosin was also observed. The gene is not essential for growth on glucose but is essential for growth on oleic acid and methanol. The role of PAS5 in peroxisome biogenesis is discussed.
100 citations
TL;DR: In vivo, mice receiving AlOx‐loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in mice receiving unloaded cells and similar amounts of meethanol, suggesting this enzyme may add an important contribution to the detoxification protocol against meetinghanol poisoning.
Abstract: Alcohol oxidase (AlOx) from Pichea pastoris (a methylotrophic yeast) was encapsulated into human and murine erythrocytes up to 2 units/ml of packed cells This enzyme has a much higher affinity for methanol than for ethanol, thus making the loaded erythrocytes useful cellular bioreactors able to catabolize methanol Enzyme-loaded erythrocytes showed an increased rate of the hexose-monophosphate-shunt activity and a significant methaemoglobin production However, the in vivo survival of these cells does not seem to be significantly affected by methanol catabolism In vivo, mice receiving AlOx-loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in mice receiving unloaded cells and similar amounts of methanol Thus AlOx-loaded erythrocytes may add an important contribution to the detoxification protocol against methanol poisoning
39 citations
TL;DR: The Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase is cloned and sequenced and expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter.
33 citations
TL;DR: A recombinant Saccharomyces cerevisiae, expressing and secreting rice α-amylase, converts starch to ethanol and a yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.
Abstract: Conversion of Starch to Ethanol in a Recombinant Saccharomyces cerevisiae Strain Expressing Rice α-Amylase from a Novel Pichia pastoris Alcohol Oxidase Promoter
31 citations
TL;DR: Three strains of methylotrophic yeasts, Candida boidinii, Pichia angusta and PichIA Pastoris, were studied for their capacity to grow on methanol in deuterated media and growth rates showed that water deuteration was the major limiting factor.
Abstract: Three 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.
17 citations
Patent•
29 Jul 1993
TL;DR: In this article, a method for producing Bacillus toxin polypeptides by culturing methylotrophic yeast cells under conditions that the gene(s) is/are transcribed is provided.
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 cells. 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.
11 citations
Journal Article•
10 citations
Patent•
06 Jan 1993TL;DR: An improved process for preparing glyoxylic acid comprising using the enzyme glycolate oxidase in the form of permeabilized microbial cell transformants selected from Aspergillus nidulans, Hansenula polymorpha, Pichia pastoris and Escherichia coli to oxidize glycolic acid with oxygen in an aqueous solution that includes an amine is described in this article.
Abstract: An improved process for preparing glyoxylic acid comprising using the enzyme glycolate oxidase in the form of permeabilized microbial cell transformants selected from Aspergillus nidulans, Hansenula polymorpha, Pichia pastoris and Escherichia coli to oxidize glycolic acid with oxygen in an aqueous solution that includes an amine.
TL;DR: In this article, the long-time stability of enzyme electrodes consisting of immobilized enzyme and an oxygen probe was examined at 30°C and 15∼20°C (the sensitivity was 60% of that found at 30-C).
Abstract: The long-time stability of enzyme electrodes consisting of immobilized enzyme and an oxygen probe was examined at 30°C and 15∼20°C (the sensitivity was 60% of that found at 30°C). The lactate (based on oxidase from Pediococcus sp.) and the alcohol (based on oxidase from Pichia pastoris) electrodes were stable for more than 14 days at 15∼16°C, but for 2∼3 days at 30°C. As to the pyruvate and the hypoxanthine electrodes, the rapid loss of the stability was prevented by reducing the operational temperature from 30°C to 18∼20°C.
Patent•
25 Mar 1993
TL;DR: In this article, two novel genomic clones, ZZA1 and ZZA2, were isolated which encode for Pichia pastoris alcohol oxidase isozymes and were used to contol the expression of other heterologous proteins in multiple yeast species.
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 AOX15' 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 5' flanking region. A yeast strain containing the ZZA1-OS103 fusion and secreting biologically active α-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.