Showing papers on "Pichia pastoris published in 1992"
TL;DR: The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification ofpas mutants than is possible in other organisms.
Abstract: We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotropic strains with an essentialiy wild type genetic background, plasmids that act as Escherichia coli–P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.
215 citations
TL;DR: The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.
Abstract: We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis.
110 citations
Patent•
24 Apr 1992
TL;DR: A process for the production of HSA in Pichia pastoris cells comprising cultivating pichia pastorsis cells capable of expressing HSA at a pH of about 5.7 to about 6.4 contemporaneously with the expression was described in this article.
Abstract: A process for the production of HSA in Pichia pastoris cells comprising cultivating Pichia pastoris cells capable of expressing HSA at a pH of about 5.7 to about 6.4 contemporaneously with the expression of HSA.
88 citations
TL;DR: Northern analysis showed that the AOD1 gene transcript was induced by methanol, but was not detected when cells were grown on ethanol or glucose, thus, as in ascosporogenous methylotrophic yeasts, A OD1 gene expression in C. boidinii appears to be controlled at the RNA level.
78 citations
TL;DR: The use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2AβPP, a secreted isoform of the Alzheimer's amyloid β-protein precursor that contains the Kunitz-type protease inhibitor (KPI) domain, is described.
74 citations
40 citations
TL;DR: Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys- leu-COOH peroxisomal targeting signal intoperoxisomes.
Abstract: This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.
35 citations
TL;DR: Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.
Abstract: The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22 466 daltons. The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coli. Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.
21 citations
TL;DR: In this article, three-dimensional alcohol oxidase crystals of space group P21 from the methylotrophic yeast Pichia pastoris grown in vitro were fragmented and studied by electron microscopy.
6 citations
TL;DR: An alcohol sensor based on the use of chemically permeabilized yeast cells was constructed and produced the highest sensitivity for methanol; the sensitivity for ethanol was about half that for meethanol and the stability and other characteristics of this new alcohol sensor are discussed.
Abstract: Chemical permeabilization of the yeast Pichia pastoris NRRL Y-11430 with 1.0% glycine and 0.1 % Triton X-100 does not release intracellular alcohol oxidase but alters the cell envelope structure, allowing alcohol oxidase activity to be detected inside the cells (Naglak, 1990). An alcohol sensor based on the use of chemically permeabilized yeast cells was constructed. The sensor response time was within 2 min. The assay time was less than 30s by the initial rate method. The calibration curve for ethanol analysis was linear in the range 5–65 mg/L. The sensor response was almost unaffected by pH changes within the range pH 5–9. This alcohol sensor produced the highest sensitivity for methanol; the sensitivity for ethanol was about half that for methanol. The stability and other characteristics of this new alcohol sensor are also discussed in this work.
5 citations
Patent•
17 Sep 1992
TL;DR: In this article, two linear DNA plasmids containing the autonomous replication sequence (ARS) and capable of self-maintenance as extra chromosomal elements are provided, which are useful in a vector for selfmaintenance in Pichia pastoris and also useful as plasmid vectors which can be shuttled between yeast and bacteria.
Abstract: Two novel linear DNA plasmids are described. Also, novel fragments of the plasmids containing the autonomous replication sequence (ARS), and thus capable of self-maintenance as extra chromosomal elements are provided. These novel DNA sequences of the present invention are useful in a vector for self-maintenance in Pichia pastoris and are also useful as plasmid vectors which can be shuttled between yeast and bacteria.
Patent•
06 Jan 1992
TL;DR: In this article, an improved expression cassette, vectors and a process for the secretion of HSA in Pichia pastoris cells was presented, in which a 5' regulatory region with 5' end and a 3' end selected from the group consisting of the PICHIA pastoris AOX1 5' and DAS1 5'.
Abstract: A novel expression cassette, vectors and process for the secretion of HSA in Pichia pastoris cells. In accordance with the present invention there has been discovered an improved expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to; b) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within about 11 deoxyribonucleotides of the 5' end of said HSA structural gene; and operably linked to c) a 3' termination sequence. In accordance with the present invention there has also discovered Pichia pastoris cells transformed with an improved expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to; b) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within about 11 deoxyribonucleotides of the 5' end of said HSA structural gene; and operably linked to c) a 3' termination sequence. In a further embodiment of the present invention, there has also discovered a process for the secretion of HSA from transformed Pichia pastoris cells comprising a) transforming a Pichia pastoris cell with at least one vector having at least one expression cassette comprising i) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably to ii) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene said HSA structural gene; and operably linked to iii) a 3' termination sequence; and then b) culturing the resulting transformed Pichia pastoris suitable conditions to obtain the secretion of HSA.
Patent•
23 Oct 1992
TL;DR: In this paper, a modified AOX2 promoter is supported on a plasmid bonded with a foreign protein (e.g. human serum albumin), which is inserted into a yeast group to effect the transformation of the yeast.
Abstract: PURPOSE:To obtain the subject plasmid capable of producing useful foreign protein at high level by depleting or substituting a part of a base sequence of a natural-type AOX2 promoter or adding a new base sequence to the above sequence and supporting the obtained promoter on a plasmid. CONSTITUTION:A natural-type AOX2 promoter of Pichia pastoris GTS115 (a kind of methanol-assimilating yeast) is cloned and the promoter is treated with restriction enzymes to deplete a part of the base sequence or substituting a part of the base sequence with another base sequence or adding a new base sequence to the original sequence. The obtained modified AOX2 promoter is supported on a plasmid bonded with a foreign protein (e.g. human serum albumin) to form a recombinant plasmid, which is inserted into a yeast group to effect the transformation of the yeast. The transformant is cultured in a medium to produce the desired foreign protein at a high production level.
TL;DR: In this paper, the methylotrophic yeast, Pichia pastoris, was used to develop a control strategy for the regulation of methanol concentration in high cell density yeast fermentations.