Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan1
Author for correspondence: Yasuyoshi Sakai. Tel: +81 75 753 6455. Fax: +81 75 753 6385. e-mail: ysakai{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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Keywords: formaldehyde oxidation pathway, gene cloning, gene disruption, energy generation, formaldehyde detoxification
Abbreviations: AOD, alcohol oxidase; DHAS, dihydroxyacetone synthase; FDH, formate dehydrogenase; FLD, glutathione-dependent formaldehyde dehydrogenase
b The GenBank accession number for the sequence reported in this paper is AB085186.
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INTRODUCTION |
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Formaldehyde is a highly reactive compound that has a toxic effect on all organisms through its nonspecific reactivity with proteins and nucleic acids (Feldman, 1973 ; Grafstrom et al., 1983
). Since formaldehyde is located at the branching point of the assimilation and dissimilation pathways, proper maintenance of the formaldehyde level is an important factor for efficient C1 metabolism. The dissimilatory formaldehyde oxidation pathway is thought to play a significant role in the detoxification of formaldehyde.
There have been some discrepancies in reports regarding the ability of FDH-defective mutants of methylotrophic yeasts to grow on methanol. Bystrykh et al. (1988) reported that an FDH-deficient mutant of Hansenula polymorpha could not grow on methanol. On the other hand, Sibirny et al. (1990)
reported that both FLD- and FDH-defective mutants of H. polymorpha could grow on methanol, and suggested that neither FLD nor FDH was essential for the energy supply for growth on methanol. Recently, a Pichia pastoris FLD-defective mutant was reported to be unable to grow on methanol (Shen et al., 1998
). We think that these discrepancies are due to the following. (1) The previous studies used mutants derived via chemical mutagenesis, and these mutant strains could exhibit leaky phenotypes. (2) Both batch and chemostat culture conditions were confusingly used for the growth analyses, leading to misevaluation of the growth ability of the mutants.
We have been using Candida boidinii as a model organism to study the physiological role of methanol-metabolizing enzymes (Horiguchi et al., 2001 ; Sakai et al., 1996
, 1997
, 1998
). One of the merits of using C. boidinii is the ease with which gene-disrupted strains can be derived (Sakai & Tani, 1992a
). Previously, we showed that an enzyme involved in the formaldehyde oxidation pathway, FDH, plays not an essential but a significant role during growth on methanol (Sakai et al., 1997
), using an FDH1-disrupted strain of C. boidinii in combination with the methanol-limited chemostat technique. In a methanol-limited chemostat culture with a low dilution rate (0·030·05 h-1), the fdh1
strain could grow on methanol as a sole carbon source, although the growth yield was diminished compared with that of the wild-type strain (Sakai et al., 1997
). These previous results raised the possibility that the formaldehyde oxidation pathway is not essential for growth on methanol of C. boidinii, although it could contribute significantly to the energy yield during growth on methanol. In this study, we focus on another NADH-generating dehydrogenase involved in the formaldehyde oxidation pathway, FLD. The physiological role of FLD was investigated through cloning, gene disruption, and expression analyses of the FLD-encoding gene in C. boidinii, in combination with chemostat-culture techniques. The results obtained showed that the glutathione-dependent formaldehyde oxidation pathway is indeed necessary for growth on methanol of C. boidinii.
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METHODS |
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MI medium was used as the basal medium (Sakai et al., 1991 ), with the carbon and nitrogen sources as follows: 1·5% (v/v) methanol, 3% (v/v) glycerol, 2% (w/v) glucose, 0·76% (w/v) NH4Cl, 0·5% (w/v) methylamine hydrochloride, 0·5% (w/v) choline chloride and 1 mM formaldehyde. The initial pH of the medium was adjusted to 6·0. Cultivation was performed on a shaking incubator at 28 °C and growth was monitored by measuring optical density at 610 nm. The formaldehyde was monitored by the method of Nash (1953)
.
E. coli was grown at 37 °C in 2xYT medium supplemented, when necessary, with ampicillin (50 µg ml-1).
Protein methods and enzyme assays.
Cells were suspended in 0·1 M potassium phosphate buffer, pH 7·0, and then transferred to a 2 ml Eppendorf tube containing an equal volume of 0·5 mm zirconium beads. The tube was shaken vigorously for 30 s with a mini-beadbeater model 693 (Biospec Products), and then chilled on ice for 30 s. This procedure was repeated six times, and the cell debris was removed by centrifugation at 16000 g for 5 min at 4 °C. The resultant supernatant was immediately used for enzyme activity assays. FLD activity was measured by determining the rate of NADH formation at 340 nm at 30 °C as described previously (Schutte et al., 1976 ). One unit of enzyme activity was defined as the amount of enzyme which produced 1 µmol NADH min-1. Protein was determined by the method of Bradford (1976)
with a protein assay kit (Bio-Rad) and bovine serum albumin as the standard. SDS-PAGE was performed with a polyacrylamide slab gel and a Tris/glycine buffer system as described by Laemmli (1970)
.
Purification of FLD.
Methanol-grown cells of C. boidinii were harvested and washed with 50 mM Tris/HCl buffer (pH 8·5). The cells were disrupted with a 200M Insornator (KUBOTA), and then the cell debris was removed by centrifugation at 20000 g. The resultant supernatant was applied to a DEAE-Sephacel column (5·0x15 cm) equilibrated with 50 mM Tris/HCl buffer (pH 8·5). After washing of the column with the same buffer, elution was carried out with a gradient of 0 to 0·5 M KCl in 50 mM Tris/HCl buffer (pH 8·5). The active fractions were pooled, concentrated, and subsequently saturated with 3·0 M ammonium sulfate. The enzyme was then applied to a Butyl-Toyopearl column (2·2x20 cm) equilibrated with 3·0 M ammonium sulfate in 50 mM Tris/HCl buffer (pH 8·5). After washing of the column with the same buffer, elution was performed with a gradient of 3·0 to 0 M ammonium sulfate. The active fractions were pooled, concentrated, and subsequently applied to a Superdex 200 column (1·6x60 cm) equilibrated with 50 mM Tris/HCl buffer (pH 8·5).
Determination of partial amino acid sequences.
The purified enzyme (1·2 mg) was digested with Achromobacter lysyl endopeptidase at an enzyme:substrate ratio of 1:200 for 12 h at 37 °C in 0·1 M ammonium bicarbonate, and the resulting peptide mixture was separated by reverse-phase HPLC on a column of Cosmosil 5C18-AR300 (4·6 mmx25 cm; Nacalai Tesque). Gradient elution was performed at 0·5 ml min-1 with 0·06% trifluoroacetic acid in water as solvent A and 0·052% trifluoroacetic acid in 80% acetonitrile as solvent B. The amino acid sequences of the amino-terminal regions of the purified enzyme and peptides were determined with a protein analyser (Applied Biosystems model 4701A) on an on-line HPLC apparatus (model 120A).
DNA methods.
Yeast DNA was purified by the method of Cryer et al. (1975) or Davis et al. (1980)
. Southern analysis was performed essentially as described previously (Yurimoto et al., 2000a
). Transformation of C. boidinii TK62 (ura3) was performed by the modified lithium acetate method, as described previously (Sakai et al., 1993
). pBluescript II SK+ and pBluescript KS- were from Stratagene, pT7Blue was from Novagen and pUC118 was from New England BioLabs. DNA was sequenced with a 7-deaza sequencing kit (Thermo sequence fluorescent labelled primer cycle sequencing kit) from Amersham Pharmacia Biotech and a DNA sequencer model DSQ-2000L from Shimadzu.
Cloning of the C. boidinii FLD1 gene.
PCR was carried out to obtain a DNA probe for screening the gene library via colony hybridization. The oligonucleotide primers were designed on the basis of internal partial amino acid sequences determined from purified FLD (Fig. 1a, LF-1 to LF-6). According to the amino acid sequences YTPECREC from LF-3 and AFHDMHAG from LF-4, a forward primer (primer 1; a 192-fold redundant 23-mer [5'-TAYACHCCWGAAWSIMGYGAAGC-3']) and a reverse primer (primer 2; a 144-fold redundant 23-mer [5'-CCDGCRTGCATRTCRTGRAADGC-3']) were designed and synthesized. The PCR reaction mixture consisted of 0·35 µg C. boidinii S2 genomic DNA as the template, 0·5 µg of each mixed primer, 0·2 mM dNTPs, 50 mM KCl, 10 mM Tris/HCl buffer (pH 8·3), 1·5 mM MgCl2, 0·001% (w/v) gelatin and 2·5 U Ex Taq DNA polymerase (Takara Shuzo) in a total volume of 100 µl. PCR was performed with a Perkin-Elmer model 480 DNA thermal cycler under the following temperature profile conditions: denaturation, 95 °C, 1 min; annealing, 56 °C, 0·5 min; extension, 72 °C, 1·5 min; for 30 cycles. The amplified 0·9 kb PCR fragment was ligated to the pT7Blue vector and then introduced into E. coli JM109 cells. The 0·9 kb NdeIBamHI fragment of the recombinant plasmid was gel-purified and used as a probe for hybridization experiments. The partially Sau3AI-digested genomic DNAs were ligated into BamHI-digested and dephosphorylated pUC118. To construct a Sau3AI gene library, E. coli JM109 cells were transformed with the resulting ligation mixture. Colonies formed on the master plates were transferred to Biodyne nylon membranes (Pall Bio Support). After lysis of E. coli and binding of the liberated DNA to the nylon membranes, the blots were used for colony hybridization by the Church and Gilbert hybridization method (Church & Gilbert, 1984
). Hybridization was performed overnight at 65 °C, and then the membranes were washed three times in 0·3xSSC (1xSSC is 0·15 M NaCl plus 0·015 M sodium citrate) at the same temperature. Positive clones were picked up from the master plates and used for further studies (pFL2).
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Construction of an FLD1 gene disruption cassette and one-step gene disruption.
To remove an approximately 0·5 kb fragment including most of the coding sequence of FLD1, the 1·0 kb SacIBamHI fragment derived from pFL2 was PCR-amplified using a pair of primers, forward primer [5'-CTGgagctcGCTATTATCAGTGTATTTTAAT-3'] and reverse primer [5'-CGggatccACGACATTCTGGAGTGTA-3'], and the other part of the 2·0 kb BamHIPstI fragment derived from pFL2 was PCR-amplified using another pair of primers, forward primer [5'-CGggatccATTGACATGGACCGATGG-3'] and reverse primer [5'-AActgcagGTGCTGTGTACGGATGTT-3'] (restriction sites are shown in lower case). These two fragments were ligated to SacIPstI-digested pBluescript II SK+. The plasmid obtained was digested with BamHI, blunt-ended, and then ligated with the blunt-ended SalIXhoI fragment derived from pSPR, which contained the C. boidinii URA3 gene with repeated flanking sequences (Sakai & Tani, 1992a ). The resulting disruption vector was digested with SacI and PstI, and then used for transformation of C. boidinii strain TK62. The disruption of the FLD1 gene (yielding the fld1
strain) and loss of the URA3 gene (yielding the fld1
ura3 strain) were confirmed by genomic Southern analysis of HindIII-digested DNA from the transformants, using the 1·0 kb SacIBamHI fragment from pFL2 as a probe (Fig. 2
).
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RESULTS |
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The FLD1 ORF consisted of two exons (encoding 6 and 368 amino acids, respectively), separated by one 123 bp intron (Fig. 1b). The intron sequence contained typical yeast intron sequences, splice junctions (5'-junction, 5'-GTAAGT-3'; 3'-junction, 5'-TAG-3'), and a branch point (5'-TACTAAC-3') (Domdey et al., 1984
; Sasnauskas et al., 1992
). The P. pastoris FLD1 has been reported to have an intron at the corresponding position (Shen et al., 1998
), but H. polymorpha FLD has no such intron (Baerends et al., 2002
).
The predicted amino acid sequence of FLD from C. boidinii showed 80%, 84%, 75%, 80%, and 80% identity to those of FLDs from P. pastoris (Shen et al., 1998 ), H. polymorpha (Baerends et al., 2002
), Candida maltosa (Sasnauskas et al., 1992
), and Saccharomyces cerevisiae (Wehner et al., 1993
), respectively. These belong to a zinc-containing alcohol dehydrogenase family (Sun & Plapp, 1992
). The cysteine residues (amino acid positions 47, 109, 112, 115, 123, and 177) and histidine residue (amino acid position 69) previously found to be within the active site (Sasnauskas et al., 1992
) are also conserved in FLD from C. boidinii.
Regulation of FLD1 expression with various carbon and nitrogen sources
We predicted that FLD1 expression might be regulated not only by methanol but also by methylamine or choline, whose metabolism also yields formaldehyde.
At first, the regulation of FLD enzyme activity was studied with various combinations of carbon and nitrogen sources for the C. boidinii wild-type strain (Fig. 3). Among the carbon sources tested, when NH4Cl was used as a single nitrogen source, only methanol gave a high level of FLD activity (Fig. 3a
, column 1). Glucose-and glycerol-grown cells did not exhibit induced levels of FLD activity (columns 2 and 5). However, when methylamine or choline was used as a single nitrogen source, FLD activity was induced in glucose- or glycerol-grown cells (columns 3, 4, 6 and 7). These results suggest that FLD1 expression was induced by formaldehyde. Indeed, the addition of formaldehyde (approx. 1 mM) to the glucose/NH4Cl medium (column 3) induced FLD activity (column 8).
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Disruption of the FLD1 gene causes a severe defect in growth on FLD-inducing C and N sources
An FLD1-gene disruption vector was constructed and then introduced into the chromosome of C. boidinii TK62 (ura3) to yield the fld1 strain. Correct gene disruption was confirmed by genomic Southern analysis with HindIII-digested genomic DNA and a 32P-labelled probe. The DNA from the wild-type strain gave a single 2·5 kb band. This band shifted to 6·5 and 3·1 kb for the fld1
and fld1
ura3 strains, respectively, as expected for FLD1 disruption and subsequent deletion of the URA3 sequence caused by a homologous recombination (Fig. 2
). In addition, methanol-induced cells of the fld1
strain did not exhibit detectable FLD activity (data not shown). These results confirmed that C. boidinii contains only one gene encoding FLD.
Next, we compared the growth of the wild-type and fld1 strains on various carbon sources with NH4Cl as the nitrogen source. The growth of both strains on glucose or glycerol was similar (data not shown). In contrast, the fld1
strain could not grow on methanol as the sole carbon source (Fig. 4a
). The level of formaldehyde accumulation in the culture medium was determined. With the wild-type strain, formaldehyde accumulated in the medium up to 0·45 mM at 36 h of culture, but then rapidly disappeared. On the other hand, with the fld1
strain, the formaldehyde level gradually increased up to 0·12 mM and did not decrease until 110 h. These results indicated that formaldehyde consumption was inhibited in the fld1
strain.
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This growth defect observed for the fld1 strain was more severe than that observed for the fdh1
strain in all cases (data not shown) (Sakai et al., 1997
). In addition, this growth defect, as well as FLD activity, recovered to levels equivalent to those in the wild-type strain on the introduction of the FLD1 gene into the fld1
ura3 strain (data not shown).
FLD is essential for growth on methanol
The accumulation of formaldehyde could be minimized by the use of methanol-limited chemostat culture conditions with low dilution rates (less than about 0·05 h-1), because the supply of methanol is the rate-limiting factor for methanol metabolism under these conditions. Previously, we showed that the C. boidinii fdh1 strain could grow on methanol as a single carbon source in a methanol-limited chemostat culture, although the growth yield was only one-fourth that of the wild-type strain (Sakai et al., 1997
). Therefore, the growth of the wild-type and fld1
strains was compared under methanol-limited chemostat cultivation conditions (Fig. 5
), which were previously applied for analysis of the fdh1
strain (Sakai et al., 1997
). Both the wild-type and fld1
strains were first grown on glucose medium, and then feeding of methanol medium was started at the dilution rate of 0·05 h-1 to observe the growth at the transition state. As expected, the cell concentration of the wild-type strain became constant after about 60 h (at 3 volume changes of the working volume), suggesting that the cells had reached methanol-limited chemostat conditions. In contrast, cells of the fld1
strain were washed out upon feeding with methanol medium. A similar result was obtained with a dilution rate of 0·03 h-1 (data not shown). These results indicate that the fld1
strain, distinct from the fdh1
strain, was not able to grow on methanol even under methanol-limited chemostat conditions. We conclude that FLD is essential for growth on methanol and suggest that this enzyme plays critical roles not only in the detoxification of formaldehyde but also in generation of energy in the form of NADH.
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DISCUSSION |
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The formaldehyde oxidation pathway comprises two NADH-generating reactions catalysed by two dehydrogenases, i.e. FLD and FDH (Anthony, 1982 ). The generated NADH molecules had been assumed to be the main source of energy during growth on methanol as a single carbon source. However, the fact that both FLD-and FDH-negative mutant strains of H. polymorpha could grow on methanol under methanol-limited chemostat culture conditions raised the possibility that the main function of the formaldehyde oxidation pathway is not energy generation but the detoxification of formaldehyde (Sibirny et al., 1990
). Our present and previous studies on FLD and FDH, which involved gene disruption and careful chemostat cultures, clearly showed that there is a difference between the fld1
and fdh1
strains in their ability to grow on methanol. The fdh1
strain could survive and grow on methanol, although the growth yield of this strain was diminished (Sakai et al., 1997
). In contrast, the fld1
strain could not grow on methanol. This difference can be explained by the two physiological functions of the formaldehyde oxidation pathway, i.e. (1) generation of energy and (2) detoxification of formaldehyde. The fdh1
strain can still generate half of the amount of NADH compared to the wild-type strain through the FLD-catalysed reaction. In contrast, NADH is not expected to be generated in the fld1
strain, because this strain cannot provide formate, another substrate for NADH generation. This difference in NADH yield clearly explains the difference in growth yield between the fld1
and fdh1
strains. Regarding formaldehyde detoxification, formate is at least 10-fold less toxic than formaldehyde (Sakai et al., 1997
). Therefore, the fdh1
strain could survive and grow on methanol when formate accumulated at a significant level (Sakai et al., 1997
). But the high toxicity of formaldehyde will not permit the fld1
strain to grow on methanol.
Although FLD has two physiological roles, i.e. energy generation and formaldehyde detoxification, we speculate that FLD is more critically involved in growth on methanol through the former rather than the latter function, based on the following observations. (1) The fld1 strain could survive and grow on choline or methylamine as the nitrogen source, formaldehyde being generated during their metabolism. In these cells, the dissimilation of carbon sources may be the main source of energy generation. Therefore, yeast cells without FLD can tolerate a certain level of formaldehyde. (2) However, the fld1
strain could not survive under methanol-limited chemostat conditions with very low dilution rates (<0·05 h-1), with which the accumulation of formaldehyde within the cells is expected to be minimized. If FLD only had a formaldehyde detoxification function, the fld1
strain would grow under these chemostat conditions.
While the formaldehyde oxidation pathway functions mainly as an energy source during growth on methanol, it seems to mainly have a formaldehyde detoxification function in methylamine and choline metabolism. As shown in Fig. 4, a higher level of formaldehyde accumulation is observed in the fld1
strain than the wild-type strain when cells are grown on glucose as the carbon source and choline or methylamine as the nitrogen source. Since these cells are assumed to generate energy mainly through the glycolytic pathway, the observed growth inhibition may be due to the toxicity of accumulated formaldehyde.
The formaldehyde oxidation pathway is not only found in methylotrophs but also in non-methylotrophs, from lower prokaryotes to higher organisms (Harms et al., 1996 ). During evolution, possibly, the formaldehyde oxidation pathway was originally used for the detoxification of formaldehyde in many organisms, and thereafter the formaldehyde oxidation pathway in some yeast cells might have acquired an energy-generation function resulting in development of the C1 pathway, which is now generally found in methylotrophic yeasts.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baerends, R. J., Sulter, G. J., Jeffries, T. W., Cregg, J. M. & Veenhuis, M. (2002). Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase. Yeast 19, 37-42.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Bystrykh, L. V., Aminova, L. R. & Trotsenko, Y. A. (1988). Methanol metabolism in mutants of the methylotrophic yeast Hansenula polymorpha. FEMS Microb Lett 51, 89-94.
Church, G. M. & Gilbert, W. (1984). Genomic sequencing. Proc Natl Acad Sci USA 81, 1991-1995.[Abstract]
Cryer, D. R., Eccleshal, R. & Murmur, J. (1975). Isolation of yeast DNA. Methods Cell Biol 12, 39-44.[Medline]
Davis, R. W., Thomas, M., Cameron, J., John, T. P. S., Scherer, S. & Padgett, R. A. (1980). Rapid DNA isolation for enzymatic and hybridization analysis. Methods Enzymol 65, 404-411.[Medline]
Domdey, H., Apostol, B., Lin, R. J., Newman, A., Brody, E. & Abelson, J. (1984). Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell 39, 611-621.[Medline]
Feldman, M. Y. (1973). Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog Nucleic Acids Res Mol Biol 13, 1-49.[Medline]
Grafstrom, R. C., Fornace, A. J.Jr, Autrup, H., Lechner, J. F. & Harris, C. C. (1983). Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells. Science 220, 216-218.[Medline]
Harms, N., Ras, J., Reijnders, W. N., van Spanning, R. J. M. & Stouthamer, A. H. (1996). S-Formylglutathione hydrolase of Paracoccus denitrificans is homologous to human esterase D: a universal pathway for formaldehyde detoxification? J Bacteriol 178, 6296-6299.[Abstract]
Horiguchi, H., Yurimoto, H., Kato, N. & Sakai, Y. (2001). Antioxidant system within yeast peroxisome: biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii. J Biol Chem 276, 14279-14288.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Nash, T. (1953). The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55, 416-421.
Sakai, Y. & Tani, Y. (1992a). Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains. J Bacteriol 174, 5988-5993.[Abstract]
Sakai, Y. & Tani, Y. (1992b). Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogenous methylotrophic yeast, Candida boidinii S2. Gene 114, 67-73.[Medline]
Sakai, Y., Kazarimoto, T. & Tani, Y. (1991). Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation. J Bacteriol 173, 7458-7463.[Medline]
Sakai, Y., Goh, T. K. & Tani, Y. (1993). High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae. J Bacteriol 175, 3556-3562.[Abstract]
Sakai, Y., Saigannji, A., Yurimoto, H., Takabe, K., Saiki, H. & Kato, N. (1996). The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme. J Cell Biol 134, 37-51.[Abstract]
Sakai, Y., Murdanoto, A. P., Konishi, T., Iwamatsu, A. & Kato, N. (1997). Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline. J Bacteriol 179, 4480-4485.[Abstract]
Sakai, Y., Nakagawa, T., Shimase, M. & Kato, N. (1998). Regulation and the physiological role of the DAS1 gene encoding dihydroxyacetone synthase in the methylotrophic yeast Candida boidinii. J Bacteriol 180, 5885-5890.
Sasnauskas, K., Jomantiene, R., Januska, A., Lebediene, E., Lebedys, J. & Janulaitis, A. (1992). Cloning and sequencing analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae. Gene 122, 207-211.[Medline]
Schutte, H., Flossdorf, J., Sahm, H. & Kula, M.-R. (1976). Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. Eur J Biochem 62, 151-160.[Abstract]
Shen, S., Sulter, G., Jeffries, T. W. & Cregg, J. M. (1998). A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216, 93-102.[Medline]
Sibirny, A. A., Ubiyvovk, V. M., Gonchar, M. V., Titorenko, V. I., Voronovsky, A. Y., Kapultsevich, Y. G. & Bliznik, K. M. (1990). Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth. Arch Microbiol 154, 566-575.
Sun, H. W. & Plapp, B. V. (1992). Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J Mol Evol 34, 522-535.[Medline]
Tani, Y., Sakai, Y. & Yamada, H. (1985). Production of formaldehyde by a mutant of methanol yeast, Candida boidinii S2. J Ferment Technol 63, 443-449.
Veenhuis, M., van Dijken, J. P. & Harder, W. (1983). The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24, 1-82.[Medline]
Wehner, E. P., Rao, E. & Brendel, M. (1993). Molecular structure and genetic regulation of SFA, a gene responsible for resistance to formaldehyde in Saccharomyces cerevisiae, and characterization of its protein product. Mol Gen Genet 237, 351-358.[Medline]
Yurimoto, H., Hasegawa, T., Sakai, Y. & Kato, N. (2000a). Physiological role of the D-amino acid oxidase gene, DAO1, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii. Yeast 16, 1217-1227.[Medline]
Yurimoto, H., Komeda, T., Lim, C. R., Nakagawa, T., Kondo, K., Kato, N. & Sakai, Y. (2000b). Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii. Biochim Biophys Acta 1493, 56-63.[Medline]
Received 26 March 2002;
revised 26 May 2002;
accepted 28 May 2002.