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Yeast 14, 1399–1406 (1998)
Isolation and Sequence Analysis of the
Orotidine-5-phosphate Decarboxylase Gene (URA3) of
Candida utilis. Comparison with the OMP
Decarboxylase Gene Family
LUIS RODRIuGUEZ*†, FRANCISCO P. CHA
u VEZ†, MARIA E. GONZA
u LEZ, LILIANA BASABE AND
TANILO RIVERO
Bioindustry Division, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba
The URA3 gene of Candida utilis encoding orotidine-5-phosphate decarboxylase enzyme was isolated by
complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the
Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5-phosphate
decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of
Saccharomyces cerevisiae. The sequence presented here has been deposited in the EMBL data library under
Accession Number Y12660. 1998 John Wiley & Sons, Ltd.
  — yeast; Candida utilis; URA3; orotidine 5-monophosphate decarboxylase; transformation system
INTRODUCTION
The yeast Candida utilis is an industrially important microorganism, which is widely used for the
production of biologically useful materials, such as
glutathione, certain amino acids, enzymes and
single-cell protein. It has been utilized in largescale production of single-cell protein from
biomass-derived sugars, such as sugar molasses
and spent sulfite liquor (Boze et al., 1994; Lawford
et al., 1979). C. utilis, as well as Saccharomyces
cerevisiae and Kluyveromyces marxianus, has been
approved for use as a foodstuff by the US Food
and Drug Administration (Boze et al., 1994).
Recently, a novel strategy concerning a transformation system for C. utilis has been reported by
*Correspondence to: L. Rodrı́guez, Bioindustry Division,
Center for Genetic Engineering and Biotechnology, PO Box
6162, Havana, Cuba. Tel: (+53) 7 21 8008; fax: (+53) 7 21
8070; e-mail: yeastlab@cigb.edu.cu
†L.R. and F.P.C. contributed equally to this work.
CCC 0749–503X/98/151399–08 $17.50
1998 John Wiley & Sons, Ltd.
Kondo et al. (1995). They obtained cycloheximide
resistant transformants by using a marker gene
containing a mutated form of the ribosomal protein L41, which conferred resistance, and also used
ribosomal DNA (rDNA) fragment as a multicopy
target for plasmid integration because the marker
needs to be present in multiple copies for selection
of cycloheximide-resistant transformants.
Transformation systems based on URA3 genes
are very powerful in S. cerevisiae (Rose et al.,
1984) and other yeasts (Yang et al., 1994; and
references therein), and have also been successfully
developed in many fungi (Benito et al., 1992; and
references therein).
The organization of the pyrimidine pathway
differs between prokaryotes and eukaryotes. Most
prokaryotes have six structural genes; however, in
yeast and fungi there are only five structural genes,
with one of them (URA2 in yeast and pyr3 in
fungi) coding for a bifunctional polypeptide, which
Received 28 March 1998
Accepted 14 June 1998
. ́  .
1400
Figure 1.
has carbamyl phosphate synthase and aspartate
transcarbamylase activities (Makoff and Radford,
1978; Souciet et al., 1989). In higher eukaryotes,
the six enzymes appear to be encoded by only
three structural genes (for review, see Jones,
1980).
The present work describes the isolation and
sequence of the URA3 gene from C. utilis, which
could be used to develop an auxotrophic transformation system in this yeast.
MATERIALS AND METHODS
Strains and plasmids
Candida utilis wild-type strain NRRL Y-1084
was used as a source of genomic DNA for the
construction of a genomic library. S. cerevisiae
SEY2202 (á leu2-112, ura3-52, his4) was used in
transformation experiments. Escherichia coli
MC1061 (F araD139 Ä(ara-leu)7696 galE15
galK16 Ä(lac)X74 rpsL (Strr) hsdR2 (rk mk + )
mcrA mcrB1) was used for the genomic library and
as host for plasmid construction, and E. coli
MC1066 (F, D LAC x74, hsr, hsm, galU, galK, trip
C 9030F, LeuB600, pyrf::Tn-5) was employed to
isolate the URA3 gene. Plasmids pURA-2 and
pURA-5, containing the URA3 gene of C. utilis,
were isolated from the genomic library by complementation screening (see text). Plasmid pUT-64
was constructed by inserting a 1·9 kb EcoRI frag 1998 John Wiley & Sons, Ltd.
(A).
ment, carrying the URA3 gene from C. utilis, in
pBsArTr (a pBR322 derivative plasmid containing
the TRP1-ARS1 fragment from YRp7); and was
used in transformation experiments.
General DNA methods
Routine recombinant DNA methodology was
performed according to Sambrook et al. (1989).
High specific-activity labelling of DNA hybridization probes was carried out by random hexamer
priming (Feinberg and Vogelstein, 1983) using
[á32P]dATP (>3000 Ci/mmol, Amersham). For
Southern blot analyses, DNA was transferred to
Hybond-N membranes (Amersham) and hybridized overnight at 42C in 6SSC buffer containing 5Denhardt’s solution, 0·5% SDS, 50%
formamide and 200 ìg/ml denatured fragmented
salmon sperm DNA (Sambrook et al., 1989). Unbound probe DNA was removed by two washes in
2SSC, 0·1% SDS at room temperature and a
wash in 0·2SSC, 0·1% SDS at 65C. DNA
sequence analysis by the dideoxy-chaintermination method (Sanger et al., 1977) was performed with double-stranded DNA plasmid
templates using Sequenase (Version 2.0) Sequencing Kit (Amersham, USB), following the manufacturer’s instructions. Genomic DNAs from yeast
cells were prepared as described by Sherman et al.
(1986).
Yeast 14, 1399–1406 (1998)
  .  3 
1401
Figure 1.
(B).
Figure 1. (A) Complementation analysis of the C. utilis URA3 gene. Growth was recorded
semi-quantitatively using a scale of + + + (high), + +, +, (low). (B) Nucleotide and derived amino
acid sequences of the URA3 gene from C. utilis.
Isolation of the URA3 gene from C. utilis
To isolate the URA3 gene from C. utilis, a
genomic DNA library of C. utilis NRRLY-1084
1998 John Wiley & Sons, Ltd.
was constructed by ligation of Sau3AI
chromosomal DNA fragments (6–9 kb) into the
BamHI-digested vector pUC19, and amplified in
Yeast 14, 1399–1406 (1998)
. ́  .
1402
E. coli MC 1061. The library was transformed in
E. coli MC 1066 and 50,000 Ampr colonies were
screened for Ura + transformants in selective
medium containing no uracil.
Computer analysis
The initial database searches were performed using BLAST (Altschul et al., 1990) with
BLOSUM62 substitution matrix and profiles
search program, both using the default parameters. The profiles search method (Gribskov
et al., 1987) was used to accumulate all sequences
of the family. We also used the FASTA program
(Pearson and Lipman, 1988) for similarity searches
in databases. A consensus multiple alignment
and the phylogenetic tree calculations were done
using the ClustalW program (Thompson et al.,
1994). The phylogenetic tree was drawn using the
Treetool program (Maciukenas, 1992).
RESULTS AND DISCUSSION
Isolation of C. utilis URA3 gene
The URA3 gene from C. utilis was isolated and
characterized. DNA frgments containing the C.
utilis URA3 gene were isolated from a C. utilis
pUC19 genomic library by the ability to complement the E. coli pyrF mutation, taking into
account that the URA3 gene from S. cerevisiae
Figure 2.
1998 John Wiley & Sons, Ltd.
Yeast 14, 1399–1406 (1998)
  .  3 
complements the pyrF mutation of E. coli (Rose
et al., 1984). When this library was spread on
uracil-deficient medium, 12 independent pyrF +
colonies were isolated. Two of these clones
(pURA-2 and pURA-5) had the same 2·8 kb
genomic C. utilis insert DNA on pUC19. DNA
from both plasmids transformed E. coli MC1066 to
Ura + at a high frequency. The inserts present in
these plasmids were shown by Southern analysis of
C. utilis total DNA to be colinear to the C. utilis
and to represent a unique sequence (not shown).
One of the C. utilis URA3 gene-pUC19 recombinants (pURA-5), which contained the 2·8 kb
fragment, was subjected to further complementation analysis (Figure 1A). The 1·9 kb EcoRI fragment, obtained from pURA-5, complemented the
pyrF mutation of E. coli MC1066 and was used to
sequence the entire C. utilis URA3 gene.
As additional evidence that the C. utilis URA3
gene and not a DNA fragment with suppressor
activity had been cloned, the 1·9 kb EcoRI fragment was cloned in a pBR322 derivative plasmid
and the resulting plasmid pUT-64 was used to
transform ura3 strain S. cerevisiae SEY2202. As a
result pUT-64 complemented the ura3 mutation in
S. cerevisiae at high frequencies.
1403
Characterization of the URA3 gene of C. utilis
A total of 1179 bp of plasmid pUREc3 were
sequenced. The nucleotide sequence and deduced
amino acid sequence of the C. utilis URA3 gene are
shown in Figure 1B. The fragment sequenced
contains 306 bp upstream from the start of translation, 800 bp of protein-coding sequence and
approximately 100 bp of 3 flanking sequence. The
C. utilis URA3 gene encodes a 266 amino-acid
protein with a theoretical molecular weight of
29,283 Da.
The nucleotides immediately flanking the ATG
initiator codon (GAAAATG) correspond well
with the consensus initiation signal (A/YAA/
YAATG) reported in yeast (Kosak, 1991). The
3-untranslated region contains a putative polyadenylation signal sequence TATAAAA (consensus
AATAAAA) found in the 3 terminal regions of
most eukaryotic genes (Guo and Sherman, 1995).
Comparison with the OMP decarboxylase gene
family
Results from previous in vivo complementation
experiments indicated that the ORF sequenced
Figure 2. Multiple alignment of the OMP DCase protein family. Absolutely conserved residues are marked with (*) and high
conserved residues with (.). The sequences are: DCOP_CANUT from C. utilis (this study), DCOP_CANPA from C. parapsilosis
(Nosek, 1996b), DCOP_CANMA from Candida maltosa (Ohkuma et al., 1993), DCOP_CANAL from Candida albicans (Ernst and
Losberger, 1989), DCOP_CANBO from Candida boidinii (Sakai et al., 1992), DCOP_CANGA from Candida glabratra (Zhou
et al., 1994), DCOP_CANTR from Candida tropicalis (Cregg et al., 1990), DCOP_KLUMA from K. marxianus (Bergkamp et al.,
1993), DCOP_KLULA from Kluyveromyces lactis (Shuster et al., 1987), DCOP_SCERV from S. cerevisiae (Rose et al., 1984),
DCOP_HANPO from Hansenula polymorpha (Merckelbach et al., 1993), DCOP_HANAN from Hansenula anomala (Ogata et al.,
1992), DCOP_HANFA from H. fabianii (M. Kato et al., unpublished results), DCOP_PICST from P. stipitis (Yang et al., 1994),
DCOP_PICOH from P. ohmeri (Piredda and Gaillardin, 1994), DCOP_PICAN from Pichia anomala (Pérez et al., 1996),
DCOP_YARLY from Yarrowia lipolytica (Strick et al., 1995), DCOP_PHYBL from Phycomyces blakesleeanus (Dı́az-Minguez
et al., 1990), DCOP_RHYCI from Rhyzomucor circinelloides (Benito et al., 1992), DCOP_SCHPO from S. pombe (Grimm et al.,
1988), DCOP_SCHCO from Schizophyllum commune (Froeliger et al., 1989), DCOP_PENCH from Penicillium chrysogenum
(Cantoral et al., 1988), DCOP_MOUSE from Mus musculus (mouse) (Ohmstede et al., 1986), PYR5_HUMAN from Homo sapiens
(human) (Suttle et al., 1988), PYR5_BOVIN from Bos taurus (bovine) (Shoeberg et al., 1993), DCOP_ASPNG from Aspergillus
niger (Wilson et al., 1988), DCOP_EMENI from Aspergillus nidulans (Oakley et al., 1987), DCOP_SORMA from Sordaria
macrospora (Nowrousian, 1996), DCOP_USTMA from Ustilago maydis (Kronstad et al., 1989), DCOP_NICTA from Nicotiana
tabacum (Millar and Kunst, 1995), DCOP_ARATH from Arabidopsis thaliana (Nasr et al., 1994), DCOP_DICDI from
Dictyostelium discideum (Jacquet et al., 1988), DCOP_DROME from Drosophila melanogaster (Eisenberg et al., 1993),
PYR5_CAELE from Caenorhabditis elegans (Wilson et al., 1994), DCOP_NEUCR from Neurospora crassa (Newbury et al.,
1986), DCOP_RHINI from Rhizopus niveus (Horiuchi et al., 1995), DCOP_TRIRE from Trichoderma ressei (Berges et al.,
1990) DCOP_TRIHA from Trichoderma harzianum (Heidenreich and Kubicek, 1994), DCOP_CEPAC from Cephalosporium
acremonium (Vian and Peñalva, 1989), DCOP_ACRLO from Acremonium lolii (Collett et al., 1995), DCOP_ENDMA from
Endomyces magnusii (Nosek, 1996a), DCOP_ECOLI from E. coli (Turnbough et al., 1987), DCOP_LACLC from Lactococcus
lactis (Andersen et al., 1996), DCOP_NAGRU from Naegleria gruberi (S. P. Remillard et al., unpublished results), DCOP_SALTY
from Salmonella typhimurium (Theisen et al., 1987), DCOP_HAEIN from Haemophilus influenzae (Fleischmann et al., 1995),
PYRF_BACCL from Bacillus caldolyticus (Ghim and Neuhard, 1994), DCOP_MYCBO from Mycobacterium bovis (Aldovini
et al., 1993), DCOP_PSEAE from Pseudomonas aeruginosa (Strych et al., 1994), DCOP_MYXXA from Myxococcus xanthus
(Kimsey and Kaiser, 1992), DCOP_BACSU from Bacillus subtilis (Quinn et al., 1991).
1998 John Wiley & Sons, Ltd.
Yeast 14, 1399–1406 (1998)
. ́  .
1404
encodes the OMP DCase in C. utilis. Further
evidence for this conclusion was sought by searching for similarities between the derived Ura3
protein and OMP DCase from other organisms.
We searched all sequences of the OMP DCase
gene extracted from databases, and compared with
C. utilis Ura3 protein.
A subset of OMP DCase was previously shown
to be highly similar at the primary sequence level
(Kimsey and Kaiser, 1992). A total of 50 OMP
DCase protein sequences were found using
BLAST and FASTA programs. The set is composed of sequences from vertebrate and invertebrate animals, a cellular slime mold, a small
nematode, plants, diverse yeast and fungi, and
both Gram-negative and Gram-positive bacteria
(Figure 2). Among the 50 OMP DCase proteins
found, the most similar to C. utilis are those from
Hansenula fabianii (81% identity), Pichia stipitis
(81%), Pichia anomala (79%) and Pichia ohmeri
(76%).
We also note that the four regions well conserved among the 20 OMP DCase analysed by
Kimsey and Kaiser (1992) (A–D in Figure 2) are
extensible to all the family. Some parts of the
sequence of OMP DCase are well conserved across
species (six residues absolutely conserved and five
highly conserved). The best-conserved region
(region B) is located in the N-terminal half of
OMP DCases and is centered around a lysine
residue which is essential for the catalytic function
of the enzyme. This region contains three residues
strictly conserved among all OMP DCases.
The radial phylogenetic tree from sequences of
the OMP DCase gene family (not shown), corroborate the presence of five distinct groups of
enzymes; the first group is formed by bacterial
OMP DCases from both, Gram-positive and
Gram-negative, the second by OMP DCases from
yeast, the third from ascomycetes fungi, the fourth
basidiomycetes fungi, and the fifth included plant,
and vertebrate and invertebrate animal OMP
DCases. Yang et al. (1994) reported that all yeast
OMP DCases, except that from Schizosaccharomyces pombe, which is not closely related to the
other yeast URA3 genes, were found in only two
clusters. We found a new cluster composed of
OMP DCase from C. utilis, H. fabianii, P. anomala
and Candida parapilopsis.
In summary, we have isolated and sequenced the
gene encoding orotidine-5-phosphate decarboxylase gene (URA3) of C. utilis. We have also updated the OMP DCase gene family. As C. utilis
1998 John Wiley & Sons, Ltd.
cells have been approved by the Food and Drug
Administration for using as a foodstuff system, it is
possible now to explore the potential of the food
yeast C. utilis as a new host for heterologous
protein production.
ACKNOWLEDGEMENTS
We would like to thank Dr Edenia Paifer for her
useful collaboration. This work was supported by
research project Bio97-1, from CIGB.
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