Analysis of alcohol oxidase isozymes in gene-disrupted strains of methylotrophic yeast Pichia methanolica

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Δ and mod1Δmod2Δ strains showed a severe defect in the growth yield, although the mod2Δ strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

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Selection of suitably non-repressing carbon sources for expression of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2014.12.007 ◽

2015 ◽

Vol 120 (1) ◽

pp. 41-44 ◽

Cited By ~ 2

Author(s):

Tomoyuki Nakagawa ◽

Keishi Wakayama ◽

Takashi Hayakawa

Keyword(s):

Carbon Sources ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Pichia Methanolica ◽

Selection Of

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The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica

Applied Biochemistry and Microbiology ◽

10.1134/s0003683809020045 ◽

2009 ◽

Vol 45 (2) ◽

pp. 137-142

Author(s):

O. A. Leonovich ◽

Yu. A. Kurales ◽

T. A. Dutova ◽

E. P. Isakova ◽

Y. I. Deryabina ◽

...

Keyword(s):

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Pichia Methanolica

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

Genetics ◽

10.1093/genetics/151.4.1379 ◽

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

Molecular Biology of the Cell ◽

10.1091/mbc.e02-07-0417 ◽

2003 ◽

Vol 14 (2) ◽

pp. 786-797 ◽

Cited By ~ 42

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.

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Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

Journal of Cell Science ◽

10.1242/jcs.96.4.583 ◽

1990 ◽

Vol 96 (4) ◽

pp. 583-590

Author(s):

M. Veenhuis ◽

J.M. Goodman

Keyword(s):

Early Stage ◽

Morphological Changes ◽

Polyclonal Antibodies ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Candida Boidinii ◽

Peroxisomal Membrane ◽

Cell Induction ◽

The Matrix ◽

Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

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