Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2

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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A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

Applied and Environmental Microbiology ◽

10.1128/aem.66.10.4253-4257.2000 ◽

2000 ◽

Vol 66 (10) ◽

pp. 4253-4257 ◽

Cited By ~ 35

Author(s):

Tomoyuki Nakagawa ◽

Tatsuro Miyaji ◽

Hiroya Yurimoto ◽

Yasuyoshi Sakai ◽

Nobuo Kato ◽

...

Keyword(s):

Methyl Ester ◽

Alcohol Oxidase ◽

Pectate Lyase ◽

Methylotrophic Yeast ◽

Pectin Methylesterase ◽

Candida Boidinii ◽

Methanol Metabolism ◽

Genes Encoding ◽

Dihydroxyacetone Synthase ◽

In Cells

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

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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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Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Journal of Bioscience and Bioengineering ◽

10.1016/s1389-1723(00)80050-x ◽

2000 ◽

Vol 89 (6) ◽

pp. 522-527 ◽

Cited By ~ 17

Author(s):

Nobuo Yamashita ◽

Toru Motoyoshi ◽

Akira Nishimura

Keyword(s):

Molecular Cloning ◽

Aspergillus Oryzae ◽

Alcohol Oxidase ◽

Isoamyl Alcohol ◽

Encoding Gene

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Cloning and sequencing of the glucosyl transferase-encoding gene from converting bacteriophage X (SFX) of Shigellaflexneri

Gene ◽

10.1016/0378-1119(93)90702-5 ◽

1993 ◽

Vol 129 (1) ◽

pp. 99-101 ◽

Cited By ~ 25

Author(s):

Naresh K. Verma ◽

Donna J. Verma ◽

Pham Thi Huan ◽

Alf A. Lindberg

Keyword(s):

Cloning And Sequencing ◽

Encoding Gene

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

Journal of Bioscience and Bioengineering ◽

10.1016/s1389-1723(01)80071-2 ◽

2001 ◽

Vol 91 (2) ◽

pp. 225-227 ◽

Cited By ~ 15

Author(s):

Tomoyuki Nakagawa ◽

Yasuyoshi Sakai ◽

Hiroyuki Mukaiyama ◽

Tasuku Mizumura ◽

Tatsuro Miyaji ◽

...

Keyword(s):

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Pichia Methanolica

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A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3945-3950.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3945-3950 ◽

Cited By ~ 6

Author(s):

Harald J. Ruijssenaars ◽

Sybe Hartmans ◽

Jan C. Verdoes

Keyword(s):

Signal Sequence ◽

Fold Increase ◽

Side Chain ◽

Cloning And Sequencing ◽

Gene Encoding ◽

E Coli ◽

Mature Enzyme ◽

Locust Bean ◽

Encoding Gene ◽

Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

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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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