J. Microbiol. Biotechnol. (2012), 22(5), 642–648 http://dx.doi.org/10.4014/jmb.1111.11071 First published online February 17, 2012 pISSN 1017-7825 eISSN 1738-8872

Cloning and Characterization of the Orotidine-5'-Phosphate Decarboxylase (URA3) from the Osmotolerant Yeast Candida magnoliae

Park, Eun-Hee1, Jin-Ho Seo2, and Myoung-Dong Kim1*

1School of Biotechnology and Bioengineering, Kangwon National University, Chuncheon 200-701, Korea 2Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea Received: November 28, 2011 / Revised: January 10, 2012 / Accepted: January 11, 2012

We determined the nucleotide sequence of the URA3 gene amounts of erythritol [10, 12, 21]. A gene transformation/ encoding orotidine-5'-phosphate decarboxylase (OMPDCase) disruption system might be essential to understand the of the erythritol-producing osmotolerant yeast Candida underlying molecular mechanisms of osmotolerance and magnoliae by degenerate polymerase chain reaction and erythritol production in C. magnoliae. However, the genetic genome walking. Sequence analysis revealed the presence manipulation of C. magnoliae is limited by a lack of markers of an uninterrupted open-reading frame of 795 bp, encoding and efficient transformation methods, which implies that a 264 amino acid residue protein with the highest identity manipulation of this strain should involve the use of to the OMPDCase of the yeast Kluyveromyces marxianus. dominant drug-resistance markers. Although several dominant Phylogenetic analysis of the deduced amino acid sequence drug-resistance markers are available [33], little success revealed that it shared a high degree of identity with other has been achieved in Candida species, which frequently yeast OMPDCase homologs. The cloned URA3 gene exhibit drug resistance and show different codon usage successfully complemented the ura3 null mutation in preferences [1, 15]. , revealing that it encodes a Genetic transformation systems employing auxotrophic functional OMPDCase in C. magnoliae. An enzyme activity markers (URA3, TRP1, HIS3, and LEU2) have been developed assay and reverse transcription polymerase chain reaction for different yeasts, because transformants can be easily indicated that the expression level of the C. magnoliae selected on drop-out media [18, 27-29]. The URA3 gene URA3 gene in S. cerevisiae was not as high as that of the S. encodes orotidine-5'-phosphate decarboxylase (OMPDCase), cerevisiae URA3 gene. The GenBank accession number which catalyzes the last step in the biosynthesis of for C. magnoliae URA3 is JF521441. [30], and has been used as a useful genetic Keywords: Candida magnoliae, URA3, orotitine-5'-phosphate marker for Saccharomyces cerevisiae and other yeasts [24, decarboxylase, complementation 25, 31]. One advantage of utilizing the URA3 gene as a selectable marker in gene transformation/disruption is that wild-type cells are not able to survive in medium containing 5-fluoro-orotic acid (5-FOA), which makes it possible to Erythritol, a four-carbon sugar alcohol, is a low caloric and develop a “Ura-blaster” for repeated use or recycling of the non-cariogenic sweetener that occurs naturally in algae, URA3 gene in “pop-in” and “pop-out” applications [2, 9]. fruits, and mushrooms [5]. Most ingested erythritol is not The present work describes the isolation and sequence metabolized by the human body and is excreted unchanged analysis of the URA3 gene from C. magnoliae (CmURA3) in the urine without changing blood glucose and insulin as the first step in the construction of a host-vector tool for levels [8, 19]. It also prevents dental caries because the an auxotrophic transformation system for this osmotolerant bacteria that cause dental caries are not able to utilize yeast. In addition, we compared the deduced amino acid erythritol as a carbon source [16]. sequence of the cloned URA3 gene with those of other Candida magnoliae JH110, an osmotolerant yeast strain yeasts to gain insight into the phylogenetic position of isolated from honeycomb [10, 32], is able to produce large C. magnoliae. The functionality of the cloned gene was *Corresponding author demonstrated by complementation of a URA3-negative S. Phone: +82-33-250-6458; Fax: +82-33-241-0508; cerevisiae strain. E-mail: [email protected] 643 Park et al.

MATERIALS AND METHODS isopropanol]. The DNA was precipitated with absolute ethanol and then dissolved in TE buffer [10 mM Tris-HCl and 1 mM EDTA Strains and Culture Conditions (pH 8.0)]. Plasmid DNA was isolated and purified from E. coli C. magnoliae KFCC 10900 [11, 32] and S. cerevisiae BY4742 using the AccuPrep Plasmid Mini Extraction Kit (Bioneer, Korea). [MATa his3∆1 leu2∆0 lys2∆0 ura3∆0] were purchased from Plasmids pGEM-T (Promega, USA) and pRS314 (ATCC 77143) EUROSCARF (European Saccharomyces cerevisiae Archive for were used for general cloning and the complementation study, Functional Analysis). S. cerevisiae EH 13.15 [13] was used to prepare respectively. genomic DNA template for polymerase chain reaction (PCR) amplification of the S. cerevisiae URA3 gene (ScURA3). Escherichia Degenerate PCR and Genome Walking coli TOP10 (Invitrogen, USA) was used for plasmid DNA preparation The degenerate oligonucleotide primers CmURA3-DGF and CmURA3- and was routinely grown at 37oC. DGR (listed in Table 1) were designed based on the core consensus LB medium (0.5% bacto-yeast extract, 1% bacto-tryptone peptone, conserved regions, FEDRKF and RYQKAG, of yeast OMPDCases and 1% NaCl) supplemented with 100 µg/ml ampicillin was used [20, 22, 27, 31]. for plasmid DNA preparation. C. magnoliae was grown in YEPD PCR reactions were performed using 5 units of Diastar Taq (1% bacto-yeast extract, 2% bacto-proteose peptone, and 2% glucose) polymerase (Solgent, Korea). For the PCR, 1 µg of template DNA at 30oC for extraction of genomic DNA. S. cerevisiae cells carrying and 100 pmol of each primer were used. The reaction conditions o plasmid were grown in synthetic complete medium lacking were 5 min at 95 C followed by 35 cycles of denaturation for 1 min o o (SC-URA-) or tryptophan (SC-TRP-), containing 2% glucose. The at 95 C, annealing for 1 min at 53 C, and extension for 1 min 30 s o S. cerevisiae transformants were grown in selective medium at 30oC at 68 C. The ~500 bp amplified fragment was cloned into the to an optical density at 600 nm (A600) of 0.8 and harvested by brief pGEM-T vector by TA cloning and sequenced. The complete open centrifugation for the OMPDCase enzyme activity assay. reading frame (ORF) and promoter and terminator regions were identified by genomic walking, which was performed with the DNA Nucleic Acid Isolation and Plasmids Walking SpeedUp Kit (Seegene, Korea) according to the manufacturer’s Yeast cells in the exponential growth phase were harvested by protocol. centrifugation, disrupted by vortexing with acid-washed glass beads (Sigma-Aldrich, USA), and the clarified lysate was applied to an Sequence Analysis anion-exchange column (Qiagen, Germany) that had been equilibrated Similarity searches for nucleotide and protein sequences were performed with 4 ml of QBT buffer [750 mM NaCl, 50 mM MOPS (pH 7.0), using the Web-based BLAST algorithm of the National Center for 15% isopropanol, and 0.15% Triton X-100]. The column was Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ washed twice with 15 ml of QC buffer [1 M NaCl, 50 mM MOPS blast). The deduced amino acid sequence was obtained using the (pH 7.0), and 15% isopropanol], and the DNA was eluted with 5 ml translational tool of the Expert Protein Analysis System (ExPASY; of QE buffer [1.25 M NaCl, 50 mM Tris-HCl (pH 8.5), and 15% http://kr.expasy.org/tools/dna.html). Multiple amino acid sequences

Table 1. Primers used in this study. Name Sequence (5'-3') Comments CmURA3-DGF TRSTYAARACNCAYATYG Degenerate PCR CmURA3-DGR GAYMGNAARTTTGCHG Degenerate PCR TSP1-cmURA3-5' ACGCGAGTACTCGCCCTT Genome walking TSP2-cmURA3-5' TCTTCGAGGCCAGACACAAT Genome walking TSP3-cmURA3-5' ATGTCCGCCCATTCGGCAAT Genome walking TSP1-cmURA3-3' CATTGTGTCTGGCCTCGAAGA Genome walking TSP2cmURA3-3' GTCGAGATTGCACGCTCGAA Genome walking TSP3cmURA3-3' GTTGGCCTCGACGACAAAGGC Genome walking ScURA3-F AATTGGATCCCTAGTGATTCACAGAAGTATG pME1043 construction ScURA3-R AATTGAATTCACTAGTGATTCACAGAAGTA pME1043 construction CmURA3-F AATTGGATCCTTCAACTTTTGCAACCCGCACGC pME1044 construction CmURA3-R AATTATCGATGCGCCGCCATTTTGTGGAAA pME1044 construction RT-cmURA3-F GGAACGTGCTGCTACTCATC RT-PCR RT-cmURA3-R TCAAAAGGCCTCTAGGTTCCT RT-PCR RT-scURA3-F GGATATTGGGTCCACCGT RT-PCR RT-scURA3-R GGCCAACGATGATGACGT RT-PCR RT-scACT1-F ATGGATTCTGAGGTTGCTG RT-PCR RT-scACT1-R AAAACGGCTTGGATGGAAA RT-PCR For degenerate primers CmURA3-DGF and CmURA3-DGR, R is A or G; S is C or G; Y is C or T; M is A or C; H is A or T or C; N is A or C or G or T. THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE FROM CANDIDA MAGNOLIAE 644 were aligned using the CLUSTALW program, and the phylogenetic were used for 40 cycles of RT-PCR amplification. Aliquots of the tree was constructed with MEGA 5.0 using the neighbor-joining PCR product were separated on a 0.8% agarose gel. method. Bootstrap analysis was performed with 1,000 replicates to test the relative support for the branches in the phylogenetic tree. Codon usage analysis was performed using the Web-based tool RESULTS AND DISCUSSION Codon Usage Database (http://www.kazusa.or.jp/codon/). Isolation and Characterization of the C. magnoliae Cloning of CmURA3 and ScURA3 URA3 Gene A TRP1-marked centromere plasmid, pRS314 (4.78 kb), was used Degenerate PCR primers targeting conserved regions of to express CmURA3 and ScURA3. To obtain CmURA3 and ScURA3 the URA3 gene were designed based on a multiple alignment , including their own promoter and terminator regions, DNA of published yeast URA3 gene sequences (Table 1). A PCR - CmURA3 fragments compassing the region from 200 to +200 bp of product of approximately 0.5 kb was amplified (data not and ScURA3 were amplified by PCR using the primers listed in o shown) at an annealing temperature of 53 C, TA-cloned, Table 1. PCR products of the expected sizes were digested with appropriate restriction enzymes. The restriction enzymes BamHI and and then sequenced. Alignment of the DNA and deduced EcoRI were used to insert the PCR-amplified ScURA3 into pRS314 amino acid sequences of the cloned product with other to obtain plasmid pME1043. Plasmid pME1044 contained a 1.2 kb yeast URA3 homologs suggested that the cloned product fragment of CmURA3 between the BamHI and ClaI sites of pRS314. contains a conserved sequence of the yeast URA3 gene [14, 22]. Based on the cloned gene sequence, six gene- Transformation specific primers listed in Table 1 were designed to further S. cerevisiae BY4742 (ura3 null strain) was transformed by the isolate the 5' and 3' regions of the CmURA3 gene. The lithium acetate method [4]. E. coli cells were transformed as described entire nucleotide sequence of the CmURA3 gene, including previously [26]. 200 bp of the 5'- and 3'-untranslated regions, was obtained after genome walking and sequence assembly, and was Complementation of a S. cerevisiae ura3 Null Strain deposited in GenBank (Accession No. JF521441). S. cerevisiae BY4742 transformants harboring plasmids pRS314, The CmURA3 gene consists of an open reading frame pME1043 (pRS314-ScURA3), and pME1044 (pRS314-CmURA3) - - (ORF) of 795 bp and encodes a putative 264 amino acid were grown in selective medium (SC-URA or SC-TRP ) for overnight - polypeptide. The estimated molecular mass and pI of this and diluted to an A600 of 0.2 with sterile SC-URA medium. Then, 5-fold serial dilutions were prepared, and 20 µl of each dilution was protein are 28.7 kDa and 5.8, respectively. Sequence analysis spotted onto a SC-URA- plate. The plates were incubated at 30oC, also indicated that the CmURA3 gene is not interrupted by and cell growth was assessed after 3 days. an intron, supported by cDNA nucleotide sequence data (data not shown). Preparation of Cell Extracts Analysis of codon usage indicated that there is a bias in A single colony of S. cerevisiae was grown in selective medium at o most codons (G/C: 64.5%; A/T: 35.5%), which might be 30 C to an A600 of 0.8, harvested by brief centrifugation, and then related to the nucleotide composition of the C. magnoliae washed twice with 0.85% NaCl solution. Cells were broken by genome. In particular, codons UCA, UAG, UGA, CUA, vortexing them with acid-washed glass beads (Sigma-Aldrich) in CCA, CAA, CGA, AUA, AAU, AGA, AGG, GUA, and 1 ml of MOPS buffer (1.5 mM, pH 7.2) for 2 min. Cell homogenates o GGA were not used at all. There was also strong bias in the were centrifuged at 15,000 ×g for 20 min at 4 C, and the supernatants were used as cell extracts. Cell extracts were prepared from three wobble position [3] of most codons (G/C: 84.5%; A/T: independent cultures. Protein concentrations were determined by the 15.5%). Bio-Rad assay using bovine serum albumin as the standard. Putative TATA elements [6] in the 5'-untranslated region were found -288 and -294 bp upstream from the expected OMPDCase Enzyme Activity Assay initiation codon. The nucleotides flanking the ATG initiation OMPDCase enzyme activity was assayed as described previously codon were GCACGCATGG, which does not correspond [17]. One unit of OMPDCase activity was defined as the amount of well with the consensus translation initiation signal reported enzyme forming 1 µmole of -5'-monophosphate per hour at in yeast [7]. In the 3'-untranslated region, two putative sequences o 25 C. The reaction mixture contained 1.5 mM MOPS buffer (pH (TTAAAAA) presumably involved in polyadenylation were 7.2), 0.1 mM EDTA, 1 mM 1,4-dithiothreitol (DTT), and protein observed 33 and 90 bp downstream of the TAA stop codon. extract. A molecular extinction coefficient for uridine-5'-monophosphate -1 The consensus sequence of the polyadenylation signal at 280nm = -1,650 cm was used. found in most eukaryotic genes is AATAAAA or AAAAAA RNA Isolation and RT-PCR [5], although several variations are possible. Total RNA was prepared using TRIzol reagent (Invitrogen). RT- Sequence alignment with other homologous OMPDCases PCR was performed using the SuperScript One-Step RT-PCR with indicated that the deduced 264 amino acid protein of C. the Platinum Taq system (Invitrogen) according to the manufacturer’s magnoliae OMPDCase has the typical structure of a instructions. Total RNA (1 µg) and the primers listed in Table 1 member of the OMPDCases, with four strongly conserved 645 Park et al.

Fig. 1. Multiple alignment of yeast OMPDCases amino acid sequences. Fully and strongly conserved residues are marked in black and grey, respectively. Conserved regions to which degenerate primers were designed are marked with asterisks. The numbers on the left and right indicate the positions of the amino acids. Candida magnoliae (GenBank Accession No. JF521441); (AAF13298); Candida dubliniensis (CAC27824); Candida glabrata (P33283); Candida maltosa (BAA02215); Candida tropicalis (ABX90091); Debaryomyces hansenii (AAK54442); Kluyveromyces marxianus (P41769); Pichia jadinii (CAA73209); Shefferomyces stipitis (AAA65978); Saccharomyces cerevisiae (AAB64498); Yarrowia lipolytica (AAA85392). THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE FROM CANDIDA MAGNOLIAE 646 domains and a lysine residue as the active site (Fig. 1). The that the URA3 gene from C. magnoliae complements the OMPDCase of C. magnoliae shared significant identity ura3∆ mutation in S. cerevisiae. However, CmURA3 did with other homologs, with highest homology to the not complement the ura3∆ mutation as efficiently as ScURA3. Kluyveromyces marxianus OMPDCase (70.4%), followed OMPDCase enzyme activity assays for the S. cerevisiae by those of S. cerevisiae (67.0%), C. glabrata (66.4%), transformants also indicated that the URA3 gene from C. and Debaryomyces hansenii (64.8%). magnoliae could recover the OMPDCase- phenotype of The alignment also revealed that several stretches of the the S. cerevisiae ura3∆ null strain (Fig. 3B). Consistent sequences are highly conserved among fungal OMPDCases: with the results shown in Fig. 3A, the OMPDCase enzyme FEDRKFADIG at amino acid positions 90-100, GQQYRT activity of S. cerevisiae transformed with ScURA3 was at 213-218, and IIVGRG at 230-235 in the sequence of 3.7-fold higher than that of the S. cerevisiae harboring C. magnoliae [22, 27, 31]. Based on the crystal structure of CmURA3, which was also supported by RT-PCR analysis. OMPDCase from S. cerevisiae [23], Asp37, Tyr217, and Transcript levels of CmURA3 in S. cerevisiae were notably Arg235 interact with substrate, whereas Asp91, Lys93, and lower than those of ScURA3, which is in good agreement Asp96 are thought to be involved in the catalytic reaction with the results shown in Fig. 3A and 3B (Fig. 3C). Based [17]. All these residues reported in S. cerevisiae were on the OMPDCase enzyme activity assay and RT-PCR conserved in the sequence of C. magnoliae OMPDCase, analysis results, we concluded that the differences in codon indicating that the cloned gene is the URA3 gene of C. usage preferences and amino acid sequences resulted in magnoliae. The similarity of the C. magnoliae OMPDCase to other yeast OMPDCases was further revealed by phylogenetic analysis (Fig. 2). The dendrogram constructed by the neighbor- joining method indicates that C. magnoliae OMPDCase is more closely related to the OMPDCases of other yeast than that of S. cerevisiae.

Complementation of the S. cerevisiae ura3∆ Strain Plasmid pRS314 was used to express C. magnoliae URA3 and S. cerevisiae URA3 in the S. cerevisiae ura3∆ strain. Growth of S. cerevisiae transformants was observed in SC- URA- plates. As shown in Fig. 3A, the S. cerevisiae ura3∆ strain harboring pRS314 (vector) was not able to grow in SC-URA- plates. However, S. cerevisiae transformants harboring the plasmids pME1043 (pRS314-ScURA3) and pME1044 (pRS314-CmURA3) successfully grew in SC- URA- medium. These complementation results indicate

Fig. 3. Growth phenotype assay (A), OMPDCase enzyme activity assay (B), and RT-PCR analysis of URA3 gene expression in a S. cerevisiae ura3 null mutant (C). Fig. 2. Phylogenetic relationships of C. magnoliae OMPDCase S. cerevisiae ura3 cells expressed CmURA3 and ScURA3 from plasmids to OMPDCases from different yeasts. pME1043 and pME1044, respectively. For growth assay, cells were The tree was constructed from an alignment of the full-length sequences of incubated at 30oC and photographed after 3 days. Results for OMPDCase OMPDCases from various yeast species using the neighbor-joining method. enzyme activity assay were obtained from three independent experiments, The numbers on the nodes correspond to the bootstrap percentages based and standard errors are shown. Total RNA (1 µg) and primers listed in on 1,000 pseudoreplicates. The bar denotes the relative branch length. Table 1 were used in a 40-cycle RT-PCR reaction. PCR products were OMPDCases are identified by their GenBank accession number in separated on a 0.8% agarose gel, and ethidium-bromide-stained. rRNA for parentheses. each sample is shown as the loading control. 647 Park et al. significantly different expression levels of the URA3 genes erythrose reductase from Candida magnoliae JH110. Microb. from these two yeast species in S. cerevisiae. Cell Fact. 9: 43. In conclusion, we successfully isolated the full-length 13. Lee, W. J., M. D. Kim, Y. W. Ryu, L. F. Bisson, and J. H. CmURA3 gene through degenerate PCR and genome walking. Seo. 2002. Kinetic studies on glucose and xylose transport in 60: We confirmed the functionality of the cloned URA3 gene Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 186-191. by complementation of ura3 auxotrophy in a S. cerevisiae 14. Li, Y., W. Shen, Z. Wang, J. Q. Liu, Z. Rao, X. Tang, et al. mutant strain. 2005. Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer. Acknowledgments Yeast 22: 423-430. 15. Lloyd, A. T. and P. M. Sharp. 1992. Evolution of codon usage This study was supported by the Basic Science Research patterns: The extent and nature of divergence between Candida Program (2011-0003791) and the Human Resource Training albicans and Saccharomyces cerevisiae. Nucleic Acids Res. 20: Project for Regional Innovation through the Korea Institute 5289-5295. for Advancement of Technology (KIAT) through the National 16. Mäkinen, K. K., M. Saag, K. P. Isotupa, J. Olak, R. Nõmmela, Research Foundation of Korea (NRF) funded by the Ministry E. Söderling, and P. L. Mäkinen. 2005. Similarity of the effects of erythritol and xylitol on some risk factors of dental caries. of Education, Science, and Technology. Caries Res. 39: 207-215. 17. Miller, B. G., M. J. Snider, R. Wolfenden, and S. A. Short. 2001. Dissecting a charged network at the active site of orotidine- REFERENCES 5'-phosphate decarboxylase. J. Biol. Chem. 276: 15174-15176. 18. Mount, R. C., B. E. Jordan, and C. Hadfield. 1996. Transformation 1. Al-Fattani, M. A. and L. J. Douglas. 2006. Biofilm matrix of of lithium-treated yeast cells and the selection of auxotrophic Candida albicans and Candida tropicalis: Chemical composition and dominant markers. Methods Mol. Biol. 53: 139-145. and role in drug resistance. J. Med. Microbiol. 55: 999-1008. 19. Noda, K. and T. Oku. 1992. Metabolism and disposition of 2. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive erythritol after oral administration to rats. J. Nutr. 122: 1266-1272. selection for mutants lacking orotidine-5'-phosphate decarboxylase 20. Park, J. M., N. S. Han, and T. J. Kim. 2007. Rapid detection activity in yeast: 5-Fluoro-orotic acid resistance. Mol. Gen. Genet. and isolation of known and putative α-L-arabinofuranosidase 197: 345-346. genes using degenerate PCR primers. J. Microbiol. Biotechnol. 3. Crick, F. H. 1966. Codon-anticodon pairing: The wobble 17: 481-489. hypothesis. J. Mol. Biol. 19: 548-555. 21. Park, S. Y., Y. W. Ryu, and J. H. Seo. 2003. Two-stage fed- 4. Gietz, R. D. and R. A. Woods. 2002. Transformation of yeast batch culture of Candida magnoliae for the production of by lithium acetate/single-stranded carrier DNA/polyethylene erythritol using an industrial medium. Kor. J. Biotechnol. Bioeng.

glycol method. Meth. Enzymol. 350: 87-96. 18: - ê 249 254. ê 5. Guo, Z. and F. Sherman. 1995. 3'-End-forming signals of yeast 22. Reinoso, C., F. Sorais, G. A. Nin o-Vega, E. Fermin án, G. San- mRNA. Mol. Cell. Biol. 15: 5983-5990. Blas, and A. Dominguez. 2005. Cloning and functional analysis 6. Harbury, P. and K. Struhl. 1989. Functional distinctions between of the orotidine-5'-phosphate decarboxylase gene (PbrURA3) of yeast TATA elements. Mol. Cell. Biol. 9: 5298-5304. the pathogenic fungus Paracoccidioides brasiliensis. Yeast 22: 7. Healy, A. and R. Zitomer. 1990. A sequence that directs 739-743. transcriptional initiation in yeast. Curr. Genet. 18: 105-109. 23. Rose, M. and D. Botstein. 1983. Structure and function of the 8. Hiele, M., Y. Ghoos, P. Rutgeerts, and G. Vantrappen. 1993. yeast URA3 gene. Differentially regulated expression of hybrid Metabolism of erythritol in humans: Comparison with glucose beta-galactosidase from overlapping coding sequences in yeast. and lactitol. Br. J. Nutr. 69: 169-176. J. Mol. Biol. 170: 883-904. 9. Kalpaxis, D., I. Zundorf, H. Werner, N. Reindl, E. Boy-Marcotte, 24. Rose, M., P. Grisafi, and D. Botstein. 1984. Structure and M. Jacquet, and T. Dingermann. 1991. Positive selection for function of the yeast URA3 gene: Expression in Escherichia Dictyostelium discoideum mutants lacking UMP synthase activity coli. Gene 29: 113-124. based on resistance to 5-fluoroorotic acid. Mol. Gen. Genet. 225: 25. Sakai, Y., T. Kazarimoto, and Y. Tani. 1991. Transformation 492-500. system for an asporogenous methylotrophic yeast, Candida 10. Kim, S. Y., Y. J. Jeon, and J. H. Seo. 1996. Analysis of boidinii: Cloning of the orotidine-5'-phosphate decarboxylase fermentation characteristics for production of erythritol by gene (URA3), isolation of uracil auxotrophic mutants, and use Candida sp. Kor. J. Food Sci. Technol. 28: 935-939. of the mutants for integrative transformation. J. Bacteriol. 173: 11. Lee, D. H., M. D. Kim, Y. W. Ryu, and J. H. Seo. 2008. 7458-7463. Cloning and characterization of CmGPD1, the Candida magnoliae 26. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning, pp. homologue of glycerol-3-phosphate dehydrogenase. FEMS Yeast 1.119-1.122, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Res. 8: 1324-1333. Spring Harbor, New York. 12. Lee, D. H., Y. J. Lee, Y. W. Ryu, and J. H. Seo. 2010. 27. Takahashi, S., R. Matsunaga, Y. Kera, and R. H. Yamada. 2003. Molecular cloning and biochemical characterization of a novel Isolation of the Cryptococcus humicolus URA3 gene encoding THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE FROM CANDIDA MAGNOLIAE 648

orotidine-5'-phosphate decarboxylase and its use as a selective 31. Van Bogaert, I. N. A., S. L. D. Maeseneire, W. D. Schamphelaire, marker for transformation. J. Biosci. Bioeng. 96: 23-31. D. Develter, W. Soetaert, and E. J. Vandamme. 2007. Cloning, 28. Tsang, P. W., B. Cao, P. Y. Siu, and J. Wang. 1999. Loss of characterization and functionality of the orotidine-5'-phosphate heterozygosity, by mitotic gene conversion and crossing over, decarboxylase gene (URA3) of the glycolipid-producing yeast causes strain-specific adenine mutants in constitutive diploid Candida bombicola. Yeast 24: 201-208. Candida albicans. Microbiology 145: 1623-1629. 32. Yu, J. H., D. H. Lee, Y. C. Park, M. G. Lee, D. O. Kim, Y. W. 29. Turakainen, H. and M. Korhola. 2005. Cloning, sequencing and Ryu, and J. H. Seo. 2008. Proteomic analysis of fructophilic application of the LEU2 gene from the sour dough yeast properties of osmotolerant Candida magnoliae. J. Microbiol. Candida milleri. Yeast 22: 805-812. Biotechnol.18: 248-254. 30. Umezu, K., T. Amaya, A. Yoshimoto, and K. Tomita. 1971. 33. Zhong, W., M. W. Jeffries, and N. H. Georgopapadakou. 2000. Purification and properties of orotidine-5'-phosphate Inhibition of inositol phosphorylceramide synthase by aureobasidin pyrophosphorylase and orotidine-5'-phosphate decarboxylase A in Candida and Aspergillus species. Antimicrob. Agents from baker’s yeast. J. Biochem. 70: 249-262. Chemother. 44: 651-653.