scholarly journals Carbon source regulation of the alcohol oxidase promoter in mutants of the methylotrophic yeast Hansenula polymorpha impaired in catabolite repression

2002 ◽  
Vol 18 (2) ◽  
pp. 155-160 ◽  
Author(s):  
O. G. Stasyk ◽  
O. V. Stasyk ◽  
A. A. Sibirny
Keyword(s):  
Carbon Source ◽  
Catabolite Repression ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Alcohol Oxidase Promoter

scholarly journals Pyruvate Carboxylase Is an Essential Protein in the Assembly of Yeast Peroxisomal Oligomeric Alcohol Oxidase

2003 ◽  
Vol 14 (2) ◽  
pp. 786-797 ◽  
Author(s):  
Paulina Ozimek ◽  
Ralf van Dijk ◽  
Kantcho Latchev ◽  
Carlos Gancedo ◽  
Dong Yuan Wang ◽  
...  
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Tricarboxylic Acid ◽  
Matrix Proteins ◽  
Growth Media ◽  
Fad Binding

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.

scholarly journals In celluloserial crystallography of alcohol oxidase crystals inside yeast cells

IUCrJ ◽  
2016 ◽  
Vol 3 (2) ◽  
pp. 88-95 ◽  
Author(s):  
Arjen J. Jakobi ◽  
Daniel M. Passon ◽  
Kèvin Knoops ◽  
Francesco Stellato ◽  
Mengning Liang ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Yeast Cells ◽  
Powder Diffraction Data ◽  
X Ray Diffraction ◽  
Protein Targets ◽  
X Ray ◽  
Serial Crystallography

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeastHansenula polymorphais reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept ofin celluloserial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

Mutant Hansenula polymorpha Strain with Constitutive Alcohol Oxidase and Peroxisome Biosynthesis

2002 ◽  
Vol 57 (9-10) ◽  
pp. 858-862 ◽  
Author(s):  
Tsonka Hristozova ◽  
Lilia Michailova ◽  
Dimka Tuneva ◽  
Velitchka Gotcheva ◽  
Angel Angelov ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Selection Procedure ◽  
Methylotrophic Yeast ◽  
Yeast Nitrogen Base ◽  
Catabolic Repression ◽  
Nitrogen Base ◽  
Ultrastructural Characteristics ◽  
Cell Plating

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains − DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol.min−1.mg−1protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.

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.

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

2006 ◽  
Vol 71 (3) ◽  
pp. 245-250 ◽  
Author(s):  
S. V. Shleev ◽  
G. P. Shumakovich ◽  
O. V. Nikitina ◽  
O. V. Morozova ◽  
H. M. Pavlishko ◽  
...  
Keyword(s):  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Purification And Characterization

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 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.

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