A methylotrophic pathway participates in pectin utilization by Candida boidinii

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.     

Dihydroxyacetone synthase is an abundant constituent of the methanol-induced peroxisome of Candida boidinii

Methylotrophic yeasts induce large peroxisomes when grown on methanol. The recent ability to stabilize and isolate these peroxisomes at pH 5.5 has led to the demonstration that two polypeptides comprise the bulk of the peroxisome of Candida boidinii, alcohol oxidase, and a 79-kDa species, determined by sodium dodecyl sulfate-polyacrylamide electrophoresis (Goodman, J.M., Scott, C.W., Donahue, P.N., and Atherton, J.P. (1984) J. Biol. Chem. 259, 8485-8493). The 79-kDa peroxisomal protein is now identified as dihydroxyacetone synthase, the first enzyme in the assimilatory pathway of formaldehyde utilization. This identification is based on several criteria: The enzyme activity is mainly in a particulate fraction at pH 5.5 but not at pH 8.0. It copurifies with alcohol oxidase and catalase on sucrose gradients. The 79-kDa protein behaves as a 135,000-kDa dimer on gel filtration, similar to the published behavior of the enzyme. The specific activity of dihydroxyacetone synthase in the pure 79-kDa preparation (3.20 units/mg of protein) is close to that reported for the purified enzyme (3.88 units/mg of protein). Antibodies against dihydroxyacetone synthase were used to show that its synthesis, induction, and assembly are similar to that of alcohol oxidase. Neither contains a detectable cleaved leader sequence and both are assembled post-translationally. The localization of dihydroxyacetone synthase to the peroxisome may influence the regulation of the two pathways of formaldehyde utilization and may protect the cell from damage by formaldehyde.     

Environmental response of yeast peroxisomes

Nutritional changes can effect either the assembly or disassembly of yeast peroxisomes. In the past decade, insights regarding the molecular mechanisms of peroxisome assembly have been gained chiefly through the cloning of the PEX genes obtained by complementation of corresponding pex mutants in several yeast strains and Chinese hamster ovary cell lines. Depletion of these peroxins (proteins encoded by PEX genes) by deletion of the corresponding genes affects peroxisomal protein import biogenesis or proliferation. To complement these studies in the field, the authors undertook an investigation of the functions of a subset of Candida boidinii peroxisomal membrane proteins (PMPs), Pex11, Pmp47, and Pmp20, by analyzing strains of C. boidnii in which the genes encoding these proteins were deleted. The authors' studies show that Pex11p is involved in peroxisome proliferation; Pmp47 plays a role in the translocation, folding, or assembly of dihydroxyacetone synthase; and Pmp20 is probably involved in methanol metabolism. In contrast to the studies on peroxisome assembly, the molecular mechanisms of peroxisome degradation remain poorly understood. To shed light on this problem, the authors isolated Pichia pastoris mutants defective in peroxisome autopathy (pag mutants). A novel, double-fluorescence method used for the characterization of wild-type cells and of pag mutants enabled us to dissect the microautophagic degradation of peroxisomes into several distinct stages. These studies show that specific PAG gene products are involved in multiple steps of the process. Future cloning and characterization of the functions of PAG genes will reveal the molecular basis of peroxisome degradation.     

Alcohol oxidase expressed under nonmethylotrophic conditions is imported, assembled, and enzymatically active in peroxisomes of Hansenula polymorpha

We have introduced into Hansenula polymorpha an extra copy of its alcohol oxidase gene. This gene which is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter is integrated in a chromosome different from the one containing the endogenous gene. Cells with the extra alcohol oxidase gene, grown on glucose or ethanol as the sole carbon source, express enzymatically active alcohol oxidase. However, other enzymes characteristic for methylotrophic growth conditions are absent or present at low levels. Most of the alcohol oxidase occurs in the octameric state and immuno- and cytochemical evidence shows that it is located in a single enlarged peroxisome per cell. Such peroxisomes show crystalloid inclusions which are lacking in the peroxisomes present in glucose grown control cells. Our results suggest that import into peroxisomes of H. polymorpha, assembly and activation of alcohol oxidase is not conditionally dependent on adaptation to methylotrophic growth conditions and that proliferation of peroxisomes is a well-programmed process that is not triggered solely by overproduction of a peroxisomal protein.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.     

Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha

The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - DTT dithiothreitol - SDS sodium dodecyl sulphate - TPP thiamin pyrophosphate - DHAS dihydroxyacetone synthase - GSH reduced glutathione     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.     

Microbiology of Wetwood: Importance of Pectin Degradation and Clostridium Species in Living Trees

Wetwood samples from standing trees of eastern cottonwood (Populus deltoides), black poplar (Populus nigra), and American elm (Ulmus americana) contained high numbers of aerobic and anaerobic pectin-degrading bacteria (10⁴ to 10⁶ cells per g of wood). High activity of polygalacturonate lyase (≤0.5 U/ml) was also detected in the fetid liquid that spurted from wetwood zones in the lower trunk when the trees were bored. A prevalent pectin-degrading obligately anaerobic bacterium isolated from these wetwoods was identified as Clostridium butyricum. Pectin decomposition by C. butyricum strain 4P1 was associated with an inducible polygalacturonate lyase and pectin methylesterase, the same types of pectinolytic activity expressed in the wetwood of these trees. The pH optimum of the extracellular polygalacturonate lyase was alkaline (near pH 8.5). In vitro tests with sapwood samples from a conifer (Douglas fir, Pseudotsuga menziesii) showed that tori in membranes of bordered pits are degraded by pure cultures of strain 4P1, polygalacturonate lyase enzyme preparations of strain 4P1, and mixed methanogenic cultures from the tree samples of wetwood. These results provide evidence that pectin in xylem tissue is actively degraded by C. butyricum strain 4P1 via polygalacturonate lyase activity. The importance of pectin degradation by bacteria, including Clostridium species, appears paramount in the formation and maintenance of the wetwood syndrome in certain living trees.     

Microbial pectic transeliminases

Pectic transeliminases, also known as pectic lyases or pectinases, are involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing. Although Aspergillus and Penicillium spp. produce pectin lyases, bacteria are the major producers of polygalacturonate lyase. The yields of pectic transeliminases are less than other pectinases. Since new applications for pectic transeliminases are emerging, an improved process for the production of these enzymes is necessary.     

An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.     

Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase

Polygalacturonate lyase is a kind of enzyme that is abundantly used in the textile industry for cotton scouring. Previously, we reconstructed the polygalacturonate lyase gene in Pichia pastoris for the expression of this enzyme. To enhance the production of polygalacturonate lyase (PGL), a combined strategy was formulated by combining online methanol control and two-stage pH control strategies. For the two-stage pH control strategy during the growth phase, the pH was controlled at 5.5, and in the induction phase different pH levels were investigated for the optimum enzyme production. During the online methanol control strategy, the different levels of methanol (v/v) were investigated for the best enzyme production at pH 5.5. These two strategies were combined together for enhanced PGL productivity, and the induction phase was divided into two stages in which methanol concentrations were maintained at different levels online. The transition phase was introduced during the induction phase instead of introducing it after the growth phase. The two-stage combination strategy was formulated on the bases of methanol consumption of cells, optimal pH, cell viability and the production of polygalacturonate lyase by P. pastoris. By using this strategy, the production was doubled compared with common conditions, and the highest polygalacturonate lyase activity reached 1,631 U/ml. This strategy proved to be very useful for the enhancement of polygalacturonate lyase production by achieving higher cell viability, alcohol oxidase activity and phosphate-related compounds of the cells during the induction phase.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Effects of macromolecular growth substrates on production of extracellular enzymes by Bacillus species in continuous culture

Bacillus species 11089 and alkalophilic Bacillus species 11203 were both capable of growth in continuous culture on macromolecular substrates, starch, soybean flour, casein, pectin, polypectate, polygalacturonate and carboxymethylcellulose (CMC) when these were used as the carbon-energy source in a mineral salts basal medium. High maximum specific growth rate (micronmax) and biomass values occurred when cells were grown on starch, soybean flour and casein, and low values on pectin, polypectate, polygalacturonic acid and CMC. Hydrolytic enzymes (protease, amylase, polygalacturonate lyase and alkaline phosphatase) were subject to regulation (induction and/or repression) depending on the nature of the growth substrate utilized. In general, high levels of enzymes were produced on soybean flour, casein and starch but low levels on CMC, pectin, polypectate and polygalacturonate.     

Properties of the enzymes catalyzing the biosynthesis of lysophosphatidate and its ether analog in cultured fibroblasts from Zellweger syndrome patients and normal controls

The activities, properties, and steady-state kinetics of the five enzymes catalyzing the synthesis of 1-acyl- and 1-alkyl-sn-glycerol 3-phosphate in the cultured skin fibroblasts from Zellweger syndrome patients and normal controls were studied in detail. Judging from their Km and Vmax values, glycerol phosphate acyltransferase (EC 2.3.1.15), acyl/alkyl dihydroxyacetone phosphate reductase (EC 1.1.1.101), and acyl coenzyme A reductase (long-chain alcohol forming), appear to be affected only slightly by the absence of peroxisomes characteristic of the Zellweger syndrome. Glycerophosphate acyltransferase also showed no differences in N-ethylmaleimide sensitivity nor in inhibition by dihydroxyacetone phosphate between these cell types. Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) and alkyl dihydroxyacetone phosphate synthase (EC 2.5.1.26) have altered activity and kinetic constants in homogenates from Zellweger syndrome fibroblasts. Dihydroxyacetone phosphate acyltransferase has similar Km (DHAP) values in both control and Zellweger syndrome cells; however, the value for the Vmax in Zellweger syndrome cells is only 6% of that found in the controls. This is interpreted as indicating that this enzyme is not defective in this disease but is simply present at a depressed level. Also, this enzyme activity has a maximum rate at pH 7.0-7.5 in the mutant cells as opposed to pH 5.4 in the controls. Acylation of dihydroxyacetone phosphate by control cell homogenate was stimulated by N-ethylmaleimide at both pH 5.7 and 7.5 whereas this activity from Zellweger syndrome cells was slightly inhibited at pH 5.7 and strongly inhibited at pH 7.5. In the absence of detergent, dihydroxyacetone phosphate acyltransferase in the Zellweger syndrome cells was much more labile to trypsin than in the control cells. Alkyl dihydroxyacetone phosphate synthase had a slightly higher Km (33 vs 17 microM) for palmitoyl dihydroxyacetone phosphate and a lower Vmax (0.07 vs 0.24 mU/mg protein) in the Zellweger syndrome cells as compared to controls. Although this is a substantial decrease in activity, it probably contributes little to the decreased rate of ether lipid synthesis in these cells. The major problem in this respect is apparently the loss of dihydroxyacetone phosphate acyltransferase activity. All of these enzymes, in both control and Zellweger syndrome cell homogenates, are sedimentable by centrifugation at 100,000g. Also, with the exception of dihydroxyacetone phosphate acyltransferase they had similar patterns of inactivation by heat in both cell types.     

Effect of temperature on the physiology and cytology of the methylotrophic yeastCandida boidinii growing in methanol-limited chemostat

All deviations from optimum cultivation temperature affect strongly the physiology and morphology of cells ofCandida boidinii strain 2 during growth in methanol-limited chemostat. The optimum cultivation temperature was 28–30 °C at which maximum cell concentration and maximum cell yield (Y _S 0.4 g/g) were achieved. At suboptimal growth temperatures the cells were rich in cell protein, RNA, alcohol oxidase (AO) and in peroxisomes. Formation of cubic peroxisomes and a 20 % decrease of budding cells in the population was observed. At supraoptimal growth temperatures (>30 °C) a sharp decrease in AO activity was accompanied by degradation of peroxisomes in the cells. The culture forms pseudomycelium: at 34 °C the cells stop growing and they are washed out of the bioreactor.     

Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.     

Production of Formaldehyde by Detergent-Treated Cells of a Methanol Yeast, Candida boidinii S2 Mutant Strain AOU-1

Treatment of cells of a methanol yeast, Candida boidinii, with the cationic detergent cetyldimethylbenzyl-ammonium chloride (Cation M2) improved the production of formaldehyde. Formaldehyde production was improved twofold with respect to the initial amount of formaldehyde and 1.61-fold with respect to the final amount of formaldehyde after a 12-h reaction under optimized detergent treatment conditions. The treatment caused formaldehyde and formate dehydrogenases to leak out of the cells more rapidly than catalase, but there was no leakage of alcohol oxidase. The improvement in formaldehyde production was considered to be due to the increased permeability of yeast cell membranes and to lower activities of formaldehyde and formate dehydrogenases in Cation M2-treated cells than in intact cells. Changes in the ultrastructure of the cells were observed upon Cation M2 treatment. Several developed peroxisomes were observed in intact cells. After Cation M2 treatment, the cells were obviously damaged, and several peroxisomes seemed to have fused with each other.     

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches’ broom disease in cacao

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.