INTRODUCTION

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Little is known about the physiological role and environmental significance of methanol-utilizing microorganisms in natural ecological systems. Methanol can be produced through the hydrolysis of pectin, which is the main constituent of primary cell walls and the middle lamellae of higher plant cells, including cells in ripening fruits, germinating seeds, developing pollen, and actively growing and degrading plant tissues. Therefore, pectin is considered to be one of the major sources of methanol in natural environments (12). Hydrolysis of the methyl ester moieties of pectin to methanol and polygalacturonate is catalyzed by pectin methylesterase (PME) (EC 3.1.1.11). Although a methylotroph is assumed to be one of the key organisms in the ecological pectin carbon cycle, no previous reports have dealt with utilization of the methyl ester moieties of pectin by methanol-utilizing organisms. Since many methylotrophic yeasts have been reported to grow on pectin as a carbon source (7), we speculated that methylotrophic yeasts are significantly involved in the ecological pectin carbon cycle. Indeed, the methylotrophic yeast Candida boidinii has been found in pectin-rich sources, including fruits and their products (olives and wine) (1).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Growth of C. boidinii S2 on pectin and polygalacturonate. Symbols: , 1% (wt/vol) polygalacturonate (DE, 0%); , 1% (wt/vol) pectin (DE, 30%); , 1% (wt/vol) pectin (DE, 60%); , 1% (wt/vol) pectin (DE, 90%) in MI medium. The initial pH and final pH of the medium were 5.7 and 5.5, respectively. (B) Growth on methanol at various concentrations. Symbols: , 0.05% (vol/vol) methanol (corresponding to the concentration of the methyl ester moiety of 1% pectin with a DE of 33%); , 0.1% (vol/vol) methanol (corresponding to a DE of 67%); , 0.15% (vol/vol) methanol (corresponding to a DE of 100%). OD660nm, optical density at 660 nm.

In a methylotrophic yeast, the first reaction in methanol metabolism is the oxidation of methanol to formaldehyde catalyzed by alcohol oxidase (AOD) (EC 1.1.3.13) (21), which is localized in peroxisomes (4). The formaldehyde produced by AOD reacts with D-xylulose 5-phosphate and produces dihydroxyacetone and glyceraldehyde 3-phosphate by dihydroxyacetone synthase (DHAS) (EC 2.2.1.3) catalysis in the methanol assimilation pathway (2, 6), and it is dissimilated to CO₂ by other enzymes, including glutathione-dependent formaldehyde dehydrogenase (FLD) (EC 1.2.1.1) and formate dehydrogenase (FDH) (EC 1.2.1.2), in the formaldehyde oxidation pathway (18). The physiological significance of these enzymes in methanol metabolism has been revealed through gene disruption analyses using C. boidinii. For example, (i) AOD is an essential enzyme for growth on methanol (8), (ii) the main role of DHAS is fixation of formaldehyde into cell constituents (15), and (iii) the physiological role of FDH has been revealed to be mainly a role in detoxification of formate rather than a role in stimulated energy generation (13). However, the physiological roles of these methanol-metabolic enzymes in pectin degradation are not known.

This study was conducted (i) to reveal the metabolic pathway for pectin degradation in the methylotrophic yeast C. boidinii (ii) and to determine the physiological roles of methanol-metabolizing enzymes and peroxisome assembly in pectin metabolism. Our results revealed that pectin is hydrolyzed to polygalacturonate and methanol by PME. Each of these products is then utilized in an independent pathway, either in conventional methanol metabolism (which requires peroxisome assembly) or in a pectin-degrading enzyme system that includes polygalacturonase (PG) (exo-PG [EC 3.2.1.67] and endo-PG [EC 3.2.1.15]), pectin lyase (PNL) (EC 4.2.2.10), and pectate lyase (PAL) (exo-PAL [EC 4.2.2.9] and endo-PAL [EC 4.2.2.2]).

	MATERIALS AND METHODS

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Yeast strains, media, and cultivation. C. boidinii S2 was used as the wild-type strain (22), and the aod1 (8), das1 (15), fdh1 (13), and pex5 (17) strains were to test growth on pectic compounds. The C. boidinii GFP-AKL strain producing GFP-PTS1 (green fluorescent protein tagged with an -AKL sequence at the carboxyl terminus that belongs to peroxisome targeting signal type 1 [PTS1]) was used to observe peroxisomal proliferation due to pectic compounds in vivo (17).

Preparation of extracellular and intracellular fractions. Yeast cells grown to the early log phase on pectic compounds were separated by centrifugation at 12,000 × g for 10 min at 4°C. Supernatants consisting of the culture media were used as extracellular fractions. The yeast cells were resuspended in 50 mM sodium phosphate buffer (pH 7.0) and were broken with a mini bead beater (Biospec Products, Bartlesville, Okla.) by using 30-s pulses with 1-min intermediate cooling periods on ice. The glass beads and cell debris were removed by centrifugation at 12,000 × g for 10 min at 4°C, and the supernatants were used as intracellular fractions for enzyme assays.

Enzyme assays. PME activity was measured by titration with 0.01 N NaOH. The reaction mixture consisted of 10 ml of 1% pectin (DE, 90%; Sigma Chemical Co.) in 50 mM acetate buffer (pH 5.0) and 1 ml of enzyme solution. Another aliquot of the sample was boiled for 10 min and used as a blank. The reaction mixture was incubated at 30°C for 60 min, and the enzyme reaction was stopped by incubation in boiling water for 10 min. The stopped reaction mixtures were titrated by using 0.01 N NaOH with phenolphthalein, and the amount of 0.01 N NaOH required was determined. One unit was defined as the amount of enzyme that released 1 µmol of carboxy groups per min.

Fluorescence microscopy. The C. boidinii GFP-AKL strain was placed on a microscope slide and examined by using the fluorescein isothiocyanate channel of an Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Plan-NEOFLUAR 100×/1 · 30 (oil) objective and Nomarski attachments and set at the fluorescein isothiocyanate channel (17). Images were acquired with a charge coupled device camera (Carl Zeiss ZVS-47DE) and a CG7 frame grabber (Scion Corp., Frederick, Md.). The number of peroxisomes per cell was determined by examining at least 350 cells in random fields.

	RESULTS AND DISCUSSION

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C. boidinii S2 was able to grow on pectic compounds. Wild-type C. boidinii strain S2 was able to grow on two pectic compounds, polygalacturonate and pectin. As the DE of pectin increased, the cell density increased (Fig. 1A). The cell density obtained on pectin with a DE of 90% was about twofold greater than that obtained on polygalacturonate (DE, 0%). This difference in growth yield between these two pectic compounds corresponds to the growth yield of C. boidinii on 0.05% methanol, on which 33% of the methyl ester moiety in pectin with a DE of 90% has been calculated to be utilized. These findings show that C. boidinii S2 has the ability to utilize the methyl ester moiety of pectin, as well as the polygalacturonate skeleton, as a sole carbon source. The C. boidinii CCY 27-37-13 strain was reported to be able to grow on a polygalacturonate medium only after adaptation to pectin (20). In contrast, C. boidinii S2 could grow on polygalacturonate medium without adaptation to pectin (Fig. 1A). Thus, the pectin metabolism system of C. boidinii S2 may be different from that of strain CCY 27-37-13, although the ability to grow on pectic compounds seems to be a general feature of this species.

PME and pectin-depolymerizing enzyme activities were induced by pectic compounds. C. boidinii S2 exhibited inducible PME enzyme activity and pectin-depolymerizing enzyme activities (i.e., PEL, PAL, and PG activities). As shown in Table 1, PME activity was not detected in cells grown on glucose medium. In contrast, PME activity was remarkably induced in cells grown on polygalacturonate or pectin. Pectin-grown cells exhibited ca. 11-fold-higher activity than the polygalacturonate-grown cells. In both cases, most PME activity was detected in the extracellular fraction (Table 1). The activities of pectin-depolymerizing enzymes (i.e., PG, PAL and PNL) was also induced in polygalacturonate or pectin medium (Table 2), and most of these activities were detected in the extracellular fraction. Although the growth of C. boidinii S2 on polygalacturonate was weak at the initial pH of the medium, pH 5.0, cells grew well when the initial pH of the medium was adjusted to 4.0 (data not shown). This may have been due to the fact that PG activity was not stable at pH 5.0 (data not shown).

Methanol-metabolizing enzymes were induced by pectic compounds. The DE affected the growth yield of C. boidinii S2 on pectin, and the data suggested that methanol produced from pectin through hydrolysis by PME was utilized by the methanol-metabolic enzymes present in C. boidinii cells. Next, we studied the regulation of the methanol-metabolizing enzymes, AOD, DHAS, FLD, and FDH by pectic compounds. As shown in Table 3, AOD, DHAS, FLD, and FDH activities were induced by pectin, although these activities were lower than those in methanol-grown cells. Also, these enzyme activities in pectin-grown cells were ca. two- to eightfold higher than those in polygalacturonate-grown cells. These results showed that both pectin and polygalacturonate could induce these methanol-metabolizing enzymes.

Pectic compounds could induce peroxisome proliferation. Since both AOD and DHAS are peroxisomal enzymes (3, 4), we examined whether pectin or polygalacturonate could induce peroxisome proliferation. To do this, a morphometric analysis using the C. boidinii GFP-AKL strain producing GFP-PTS1 (green fluorescent protein tagged with an -AKL sequence at the carboxyl terminus) was performed. When the GFP-AKL strain was grown on synthetic glucose medium, there were a few small peroxisomes (1.29 ± 0.13 peroxisomes/cell) (Fig. 2A). The numbers of peroxisomes after growth on pectin and polygalacturonate were 3.34 ± 0.31 and 2.76 ± 0.22 peroxisomes/cell, respectively (Fig. 2B and C). On the other hand, cells grown on methanol had large peroxisomes (4.8 ± 0.41 peroxisomes/cell) (Fig. 2D). The peroxisome inducers in pectin-grown cells seemed to be pectic compounds and not methanol derived from pectin through hydrolysis, based on the following observations: (i) peroxisomes were induced by polygalacturonate, which does not include methyl ester moieties (Fig. 2C), and (ii) the morphology of peroxisomes induced by pectin was different from that in methanol-grown cells (Fig. 2B and D).

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Fluorescence of GFP-AKL/wt induced by glucose (A), pectin (DE, 90%) (B), polygalacturonate (DE, 0%) (C), and methanol (D). The induced cells were visualized by Nomarski microscopy (upper micrographs) or fluorescence microscopy (lower micrographs).

Disruption of the AOD1, DAS1, FDH1, and PEX5 genes caused a defect in growth on pectin medium but not on polygalacturonate medium. To determine to what extent methanol-metabolic enzymes and peroxisome assembly were directly involved in pectin metabolism, knockout strains depleted of AOD, DHAS, and FDH (i.e., aod1, das1, and fdh1 strains), as well as the PTS1 receptor responsible for peroxisome assembly (i.e., a pex5 strain), were grown on pectin or polygalacturonate as a carbon source, and the growth of each strain was compared with that of the wild-type strain.

aod1

das1

fdh1

pex5

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Growth of the wild type (A) and the pex5 (B), aod1 (C), das1 (D), and fdh1 (E) strains on polygalacturonate (DE, 0%) () and pectin (DE, 90%) (). The initial pH of the medium was 5.0. OD660nm, optical density at 660 nm.

Mechanism of pectin assimilation in C. boidinii. In this study, C. boidinii S2 was shown to have the ability to utilize each of two pectic compounds, pectin and polygalacturonate, as a carbon source. This strain produced pectin-depolymerizing enzymes. Furthermore, utilization of methanol derived from hydrolysis of the methyl ester moiety of pectin was found to contribute significantly to the growth yield of C. boidinii on pectin, based on the following observations: (i) the cell density increased as the DE of pectin increased (Fig. 1); (ii) C. boidinii cells grown on pectin had induced levels of PME activity and activities of methanol-metabolizing enzymes (Tables 1 and 3); and (iii) the knockout strains devoid of methanol-metabolizing enzymes (the aod1, das1, and fdh1 strains) showed a severe growth defect on pectin (Fig. 3). In addition, peroxisome assembly was also found to be involved in pectin metabolism.