Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan
Correspondence
Tomoyuki Nakagawa
t-nakaga{at}bioindustry.nodai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In natural environments, plants are the major source of methanol; for example, methanol can be detected in leaf extracts and is emitted from leaves into the atmosphere (MacDonald & Fall, 1993; Nemecek-Marshall et al., 1995
). In plants, one of the major sources of methanol is pectin, which is hydrolysed to methanol and polygalacturonate by pectin methylesterase (PME) (Sakai et al., 1993
). It has been reported that many methylotrophic yeasts are able to grow on pectic compounds as sole carbon sources (Lee & Komagata, 1980
). Therefore, we believe that this pectic-growth ability of methylotrophic yeasts may be one of the clues for revealing the physiological and ecological roles of yeasts and the methanol cycle in natural environments. Indeed, the methylotrophic yeast Candida boidinii grown on pectin media exhibits the activities of pectin-metabolizing enzymes and methanol-metabolizing enzymes (Nakagawa et al., 2000
; Stratilova et al., 1998
).
In a methylotrophic yeast, the first reaction in methanol metabolism is the oxidation of methanol to formaldehyde catalysed by alcohol oxidase (AOD) (Tani et al., 1978), which is localized in peroxisomes (Goodman et al., 1984
). The methylotrophic yeast Pichia methanolica has nine AOD isozymes, AOD being the first enzyme for methanol utilization during methylotrophic growth (Gruzman et al., 1996
; Nakagawa et al., 1996
). The AOD isozymes of P. methanolica are encoded by two genes, MOD1 and MOD2 (Nakagawa et al., 1999
, 2001
; Raymond et al., 1998
). However, little is known about the physiological roles of methanol-metabolizing enzymes and AOD isozymes in pectin utilization by P. methanolica, although one of the major sources of methanol is pectin in natural environments.
This study was conducted to reveal the metabolic pathway for pectin degradation in the methylotrophic yeast P. methanolica, and to determine the physiological roles of AOD isozymes in pectin metabolism in natural environments.
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METHODS |
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Complex YPD and synthetic MI media were used for cultivation of the P. methanolica strains (Sakai et al., 1995, 1998
). The carbon source was one of the following: 1 % (w/v) glucose, 1 % (v/v) glycerol, 1 % (v/v) methanol, 1 % (v/v) oleic acid, 1 % (w/v) pectin or 1 % (w/v) polygalacturonate (Sigma). The degree of esterification (DE) of pectins from citrus fruit (Sigma) was approximately 90 %. The initial pH of the medium was adjusted to 4·0 or 6·0. Cultivation was performed aerobically at 28 °C with rotary shaking, and growth was followed by measuring OD660.
Preparation of extracellular and intracellular fractions.
Yeast cells grown on pectic compounds were separated by centrifugation at 12 000 g for 10 min at 4 °C, supernatants of the culture media being used as extracellular fractions. The yeast cells were resuspended in 50 mM sodium phosphate buffer (pH 7·0), and then disrupted with a mini bead-beater (Biospec Products) for periods of 30 s, with intermediate cooling periods of 1 min 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.
Pectin methylesterase (PME), polygalacturonase (PG), pectin lyase (PNL) and pectate lyase (PAL) activities were determined as described previously (Nakagawa et al., 2000, 2005
). One unit of PME was defined as the amount of enzyme that released 1 µmole of carboxy group per minute. One unit of PG was defined as the amount of enzyme that released 1 µmole of reducing group per minute, and reducing groups derived from polygalacturonate were measured by the method of Somogyi and Nelson (Nelson, 1944
; Somogyi, 1952
). One unit of PNL and PAL, respectively, was defined as an increase in A235 of 1·0 of the reaction mixture per minute (Ishii & Yokotsuka, 1972
).
Alcohol oxidase (AOD) (Tani et al., 1985), dihydroxyacetone synthase (DHAS) (Nash, 1953
; Yanase et al., 1995
), glutathione-dependent formaldehyde dehydrogenase (FLD) (Schütte et al., 1976
; Nakagawa et al., 2004
) and formate dehydrogenase (FDH) (Schütte et al., 1976
) activities were determined as described previously, and each activity was defined as given in the respective paper.
Protein was determined by the method of Bradford (1976) with a protein assay kit (Bio-Rad), using BSA as standard.
Electrophoresis of AOD isozymes.
For AOD-zymogram analysis of P. methanolica mutant strains, 20 µg aliquots of cell-free extracts of strains grown on pectin were subjected to non-denaturing 5 % native PAGE at 4 °C according to the method of Laemmli (1970). After electrophoresis, the polyacrylamide gels were stained by oxidation with guaiacol (Lee & Komagata, 1980
; Nakagawa et al., 1996
).
Northern analysis.
P. methanolica cells grown on or induced with several carbon sources were harvested by centrifugation at 6700 g for 10 min at 4 °C, and then total RNAs were extracted from the cells by the acid-guanidinium thiocyanate-phenol/chloroform method using ISOGEN (Nippon Gene Co.). Briefly, 10 µg aliquots of RNA samples were electrophoresed on 1·0 % agarose gels containing 20 mM MOPS buffer, 1 mM EDTA and 2·2 M formaldehyde. After electrophoresis, capillary transfer to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech) in 20x SSC (1x SSC=0·15 M NaCl plus 0·015 M sodium citrate) was performed. The DNA probe consisted of the entire coding region of MOD1 or MOD2 and was labelled with an AlkPhos DIRECT kit (Amersham Pharmacia Biotech).
Expression of green fluorescent protein (GFP).
Expression vector pMETB (Invitrogen) was used for the expression of GFP in P. methanolica. GFP-coding regions were PCR-amplified, using the GFP-ATG primer, 5'-CTCgAgATggTgAgCAAgggCgAgg-3', and GFP-SKL primer, 5'-TgTCgACTTATAATTTAgACTTgTACAgCTCgTCCATgC-3'. The obtained PCR fragment was introduced into the XhoI and SalI sites of pMETB under the PmMOD1 promoter, yielding pMET-GFP-SKL. The vector was linearized with PstI and used for the transformation of strain PMAD11. The transformant was termed strain GFP-SKL/wt.
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RESULTS AND DISCUSSION |
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Since methyl esterification of pectin affected the growth yield of P. methanolica on pectin and the yeast exhibited PME activity, it seems that methanol produced from pectin was utilized by the methanol-metabolizing enzymes in P. methanolica cells. Next, we studied the regulation of the methanol-metabolizing enzymes of P. methanolica by pectic compounds. As shown in Table 1, alcohol oxidase (AOD), dihydroxyacetone synthase (DHAS), glutathione-dependent formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) activities were detected in pectin-grown cells, although these activities were lower than those in methanol-grown cells. Also, these enzyme activities in pectin-grown cells were
1·7- to 4·3-fold higher than those in polygalacturonate-grown cells.
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Mod1p is able to function in pectic metabolism and growth, but Mod2p is not
P. methanolica grown on pectin exhibits AOD activity, but P. methanolica has AOD isozymes (Ashin & Trotsenko, 1998; Gruzman et al., 1996
; Nakagawa et al., 1996
) and two AOD-encoding genes, MOD1 and MOD2 (Nakagawa et al., 1999
; Raymond et al., 1998
). To determine to what extent the AOD genes are directly involved in pectin metabolism, AOD gene knock-out strains were grown on pectin or polygalacturonate as a carbon source, their growth being compared with that of the wild-type strain (Fig. 2
).
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Next, we attempted to identify the gene encoding the pectin-inducible AOD by zymogram analysis and Northern analysis. A cell-free extract of pectin-grown cells gave a single AOD band on zymogram analysis, the Rm value corresponding to that of Mod1p (Fig. 3A). The cell-free extracts of the mod1
and mod1
mod2
strains did not give any AOD-active band, although those of the wild-type and mod2
strains each gave a single band corresponding to Mod1p (Fig. 3A
). In addition, MOD1- and MOD2-gene expression was followed at the mRNA level. The expression of MOD1 was detected by pectin, but the MOD2-gene expression detected by pectin was much lower than that of MOD1 (Fig. 3B
). Moreover, disruption of MOD1 caused a growth defect on pectin medium. These findings indicated that Mod1p is the functional AOD subunit in pectin utilization, and that the presence of Mod2p alone is not sufficient for growth on pectin. It has been reported that Mod2p exhibits a
10-fold higher Km value toward methanol compared to Mod1p (Gruzman et al., 1996
; Nakagawa et al., 1996
, 2002
). Therefore, it seems that Mod2p cannot function in pectin medium with a low methanol concentration, although the expression of MOD2 to utilize methanol derived from pectin, can be detected slightly by pectin.
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Pectin is an inducer of peroxisome proliferation
Since (i) Mod1p, which is detected in pectin-grown cells, is a peroxisomal enzyme, (ii) methanol derived from pectin is metabolized in peroxisomes, and (iii) peroxisome proliferation in C. boidinii was induced by pectin (Nakagawa et al., 2000), we examined whether or not pectin could induce peroxisome proliferation in P. methanolica. Morphometric analysis of peroxisomes was performed using P. methanolica strain GFP-SKL producing GFP-PTS1 (GFP tagged with an SKL sequence at the carboxyl terminus). When strain GFP-SKL was grown on glycerol medium, there were very small peroxisomes (Fig. 4B
). On the other hand, cells grown on pectin had large peroxisomes, like cells grown on oleic acid, which is one of the peroxisome inducers (Fig. 4A, D
), although their morphology was smaller than that of methanol-grown cells (Fig. 4C
). These findings showed that pectin is a peroxisome inducer, like methanol and oleic acid.
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From the pectic-growth data for P. methanolica and C. boidinii (Nakagawa et al., 2000; Stratilova et al., 1998
), it seems that utilization of both the methylester moiety and the polygalacturonate skeleton of pectin as carbon sources is a general feature of methylotrophic yeasts, and that methylotrophic yeasts are significantly involved in the ecological carbon cycle of pectin in natural environments.
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ACKNOWLEDGEMENTS |
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Received 16 January 2005;
revised 16 February 2005;
accepted 7 March 2005.
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