Regulation of intracellular formaldehyde toxicity during methanol metabolism of the 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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Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

Journal of Cell Science ◽

10.1242/jcs.97.1.193 ◽

1990 ◽

Vol 97 (1) ◽

pp. 193-204 ◽

Cited By ~ 1

Author(s):

J.M. Goodman ◽

S.B. Trapp ◽

H. Hwang ◽

M. Veenhuis

Keyword(s):

Oleic Acid ◽

Membrane Proteins ◽

Carbon Sources ◽

Methylotrophic Yeast ◽

Candida Boidinii ◽

Acid Oxidase ◽

General Function ◽

Amino Acid Oxidase ◽

Peroxisomal Membrane ◽

Methanol Metabolism

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 × 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are ‘substrate-nonspecific’. We speculate that they may relate to the structure, assembly or general function of the organelle.

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Methanol sensor Wsc1 and MAP kinase suppress degradation of methanol-induced peroxisomes in methylotrophic yeast

Journal of Cell Science ◽

10.1242/jcs.254714 ◽

2021 ◽

pp. jcs.254714

Author(s):

Shin Ohsawa ◽

Koichi Inoue ◽

Takahiro Isoda ◽

Masahide Oku ◽

Hiroya Yurimoto ◽

...

Keyword(s):

Methylotrophic Yeast ◽

Plant Cell Walls ◽

Peroxisome Biogenesis ◽

Plant Leaves ◽

Methanol Metabolism ◽

Methanol Sensor ◽

Key Factor ◽

Inducible Genes ◽

Hydrolysis Of ◽

Synthesis And Degradation

In nature, methanol is produced during the hydrolysis of pectin in plant cell walls. Methanol shows circadian dynamics on plant leaves to which methanol-utilizing phyllosphere microorganisms adapt. In the methylotrophic yeast Komagataella phaffii (Pichia pastoris), the plasma membrane protein KpWsc1 senses environmental methanol concentrations, and transmits the information to induce genes for methanol metabolism together with huge peroxisomes. In this study, we show that KpWsc1 and its downstream MAPK negatively regulate pexophagy in the presence of >0.15% methanol. Although KpMpk1 was not necessary for expression of methanol-inducible genes and peroxisome biogenesis, KpMpk1, KpRlm1 and a phosphatase were found suppress pexophagy by controlling phosphorylation level of KpAtg30, the key factor of pexophagy. We reveal at the molecular level how the single methanol sensor KpWsc1 commits the cell to peroxisome synthesis and degradation according to the methanol concentration, and discuss the physiological significance of regulating pexophagy for survival in the phyllosphere.

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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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Function and regulation of an aldehyde dehydrogenase essential for ethanol and methanol metabolism of the yeast, Komagataella phaffii

10.1101/2020.06.14.151365 ◽

2020 ◽

Author(s):

Kamisetty Krishna Rao ◽

Umakant Sahu ◽

Pundi N Rangarajan

Keyword(s):

Transcriptional Activation ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Ethanol Metabolism ◽

Mrna Levels ◽

Methanol Metabolism ◽

Protein Levels ◽

Komagataella Phaffii

AbstractThe genome of the methylotrophic yeast, Komagataella phaffii harbours multiple genes encoding putative alcohol dehydrogenases and aldehyde dehydrogenases (ALDs). Here, we demonstrate that one of the ALDs denoted as ALD-A is essential for ethanol metabolism. A zinc finger transcription factor known as Mxr1p regulates ALD-A transcription by binding to Mxr1p response elements (MXREs) in the ALD-A promoter. Mutations which abrogate Mxr1p binding to ALD-A MXREs in vitro abolish transcriptional activation from ALD-A promoter in vivo. Mxr1p regulates ALD-A expression during ethanol as well as methanol metabolism. ALD-A is essential for the utilization of methanol and Δald-a is deficient in alcohol oxidase (AOX), a key enzyme of methanol metabolism. AOX protein but not mRNA levels are down regulated in Δald-a. ALD-A and AOX localize to cytosol and peroxisomes respectively during methanol metabolism suggesting that they are unlikely interact with each other in vivo. This study has led to the identification of Mxr1p as a key regulator of ALD-A transcription during ethanol and methanol metabolism of K. phaffii. Post-transcriptional regulation of AOX protein levels by ALD-A during methanol metabolism is another unique feature of this study.

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GENOMIC DIVERSITY, CHROMOSOMAL REARRANGEMENTS, AND INTERSPECIES HYBRIDIZATION IN THE OGATAEA POLYMORPHA SPECIES COMPLEX

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab211 ◽

2021 ◽

Author(s):

Sara J Hanson ◽

Eoin Ó Cinnéide ◽

Letal I Salzberg ◽

Kenneth H Wolfe ◽

Jamie McGowan ◽

...

Keyword(s):

Species Complex ◽

Population Genomics ◽

Chromosomal Rearrangements ◽

Nitrate Assimilation ◽

Methylotrophic Yeast ◽

Genomic Diversity ◽

Methanol Metabolism ◽

Interspecies Hybridization ◽

Mating Type Switching ◽

Ogataea Polymorpha

Abstract The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the Ogataea polymorpha species complex, including representatives of the species O. polymorpha, O. parapolymorpha, O. haglerorum, and O. angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

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