scholarly journals Catabolite Repression of Aox in Pichia pastoris Is Dependent on Hexose Transporter PpHxt1 and Pexophagy

2010 ◽  
Vol 76 (18) ◽  
pp. 6108-6118 ◽  
Author(s):  
Ping Zhang ◽  
Wenwen Zhang ◽  
Xiangshan Zhou ◽  
Peng Bai ◽  
James M. Cregg ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Catabolite Repression ◽  
Alcohol Oxidase ◽  
Sugar Metabolism ◽  
Methylotrophic Yeast ◽  
Degradation Pathway ◽  
Hexose Transporter ◽  
Heterologous Proteins ◽  
Induced Expression ◽  
Transcript Levels

ABSTRACT In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an Δhxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

2009 ◽  
Vol 76 (4) ◽  
pp. 1062-1070 ◽  
Author(s):  
Anna Steinle ◽  
Sabrina Witthoff ◽  
Jens P. Krause ◽  
Alexander Steinbüchel
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Medium Composition ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
C Terminus ◽  
Fermentation Parameters ◽  
Optimization Of Fermentation ◽  
In Cells

ABSTRACT Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-l-arginyl-poly-l-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA 6308 and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA6308Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA6308C595S; and the combined double-mutant CphA6308Δ1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA6308 (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA6308Δ2) or three (CphA6308Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA6308. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA6308Δ1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308Δ1.

scholarly journals Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Xueyun Zheng ◽  
Yimin Zhang ◽  
Xinying Zhang ◽  
Cheng Li ◽  
Xiaoxiao Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Recombinant Proteins ◽  
Recombinant Protein Expression ◽  
Large Subunit ◽  
Protein A ◽  
Methylotrophic Yeast ◽  
Regulation Mechanism ◽  
Heterologous Proteins ◽  
Foreign Proteins

Abstract Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

scholarly journals Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50

2010 ◽  
Vol 76 (10) ◽  
pp. 3314-3324 ◽  
Author(s):  
Antonio Basanta ◽  
Beatriz Gómez-Sala ◽  
Jorge Sánchez ◽  
Dzung B. Diep ◽  
Carmen Herranz ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Enterococcus Faecium ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Secretion Signal ◽  
Expression Host ◽  
Sec System ◽  
Alcohol Oxidase Promoter ◽  
The Individual ◽  
First Time

ABSTRACT In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s ) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast.

Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris

1995 ◽  
Vol 108 (1) ◽  
pp. 25-35 ◽  
Author(s):  
D.L. Tuttle ◽  
W.A. Dunn
Keyword(s):  
Protein Synthesis ◽  
Pichia Pastoris ◽  
Carbon Source ◽  
Formate Dehydrogenase ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Intermediate Stage ◽  
Peroxisomal Enzyme ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroautophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.

scholarly journals The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris

2018 ◽  
Vol 293 (43) ◽  
pp. 16647-16660 ◽  
Author(s):  
Trishna Dey ◽  
Kamisetty Krishna Rao ◽  
Jesminara Khatun ◽  
Pundi N. Rangarajan
Keyword(s):  
Transcription Factor ◽  
Pichia Pastoris ◽  
Signaling Pathway ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Leucine Zipper ◽  
Alcohol Oxidase ◽  
Transcriptional Regulator ◽  
Methylotrophic Yeast ◽  
Methanol Metabolism ◽  
Methylotrophic Yeast Pichia Pastoris

Rtg1p and Rtg3p are two basic helix–loop–helix, retrograde transcription factors in the budding yeast Saccharomyces cerevisiae. Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast Pichia pastoris. We observed that P. pastoris Rtg1p (PpRtg1p) heterodimerizes with S. cerevisiae Rtg3p and functions as a nuclear, retrograde transcription factor in S. cerevisiae, but not in P. pastoris. We noted that P. pastoris Rtg3p lacks a functional leucine zipper and interacts with neither S. cerevisiae Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple P. pastoris metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source. PpRtg1p also had an essential role in methanol metabolism and regulated alcohol oxidase synthesis and was required for the metabolism of ethanol, acetate, and oleic acid, but not of glucose and glycerol. Although PpRtg1p could functionally complement ScRtg1p, ScRtg1p could not complement PpRtg1p, indicating that ScRtg1p is not a functional PpRtg1p homolog. Thus, PpRtg1p functions as a nuclear, retrograde transcription factor in S. cerevisiae and as a cytosolic, post-transcriptional regulator in P. pastoris. We conclude that PpRtg1p is a key component of a signaling pathway that regulates multiple metabolic processes in P. pastoris.

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