The nucleotide sequence of the
gene encoding phosphoribosyl anthranilate isomerase in yeast
was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in
was 43.5% homologous to that of
. The cloned
) complemented the
, suggesting that it encodes a functional
. A new auxotrophic marker to engineer starch-degrading yeast
is now available. The GenBank Accession No. for
Gene transformation or disruption would be a useful tool to analyze the molecular mechanisms in yeast
. Genetic transformation systems employing auxotrophic markers, such as
URA3, TRP1, HIS3,
, have been developed for different yeasts, because transformants can be easily selected on drop-out media
gene encodes phosphoribosyl anthranilate isomerase, which catalyzes the third step in tryptophan biosynthesis
and is commonly used as a selectable marker in yeasts
is the major amylolytic yeast found in starchy substrates, such as bread dough, alcoholic beverages, and rice cake starters
. Co-cultures of
are used to produce ethanol from starch
. These co-cultures produce amylase, protease, β-glucosidase, and trehalose, and have applications in food, biofuel, and pharmaceutical industries
. In our previous study, a thermotolerant
strain was isolated from
, a traditional Korean starter for rice wine fermentation
In this study, we isolated and sequenced the
as the first step in the construction of a host-vector tool for an auxotrophic transformation system for this starch-utilizing yeast. The functionality of the cloned
was demonstrated by complementation of a
ura3-52 lys2-801_amber ade2-101_ochre trp1-∆63 his3-∆020 leu2-∆1
] (Clontech, Palo Alto, CA, USA) were used.
TOP10 (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for plasmid DNA preparation and was grown at 37℃ in LB medium (5 g/l bacto-yeast extract, 10 g/l bactotryptone peptone, and 10 g/l NaCl) supplemented with ampicillin (100 mg/l). To prepare genomic DNA,
was grown in YEPD (10 g/l bacto-yeast extract, 20 g/l bactoproteose peptone, and 20 g/l glucose) at 30℃.
Degenerate oligonucleotide primers TRP3F (5’-GTN GGNGTNTTYMGNAAYCARWSN-3’) and TRP3R (5’-NGT YTCNACNCCNCCNSWNACRTC-3’) were designed based on the core consensus conserved regions VGVFRNQS and DVSGGVET
. The PCR was performed using 2.5 units of TOPsimple nTaq polymerase (Enzynomics, Daejeon, Korea). The reaction condition was 5 min at 94℃, followed by 30 cycles of denaturation for 30 sec at 94℃, annealing for 30 sec at 58℃, and extension for 30 sec at 72℃, with a final 5 min elongation at 72℃. The amplified fragment (approximate 400 bp) was cloned to the pTOP TA V2 vector (Enzynomics) and sequenced.
cells were transformed as described previously
. The complete open reading frame (ORF), promoter, and terminator regions were obtained by genomic walking, which was performed with the DNA Walking SpeedUp Kit (Seegene, Seoul, Korea) according to the manufacturer’s protocol.
Nucleotide and protein sequence similarity searches were performed using the Web-based BLAST algorithm of the National Center for Biotechnology Information (NCBI,
). Multiple amino acid sequence alignment was performed by using San Diego Supercomputer Center (SDSC) Biology Workbench (
. The C-DART program (Conserved Domain Architecture Retrieval Tool) from NCBI was used to compare the amino acid sequence of the protein with database sequences.
with its own promoter and terminator regions, approximately 300 bp of 5’ and 3’ regions of the
were amplified by PCR using SfTRP1-F (5’-AATTGAGCTCTCTGGGCCTATTGATAACTCATC-3’) and SfTRP1-R (5’-AATTGTCGACTGCGAGTTTTAGCGACA AACTTT-3’). The PCR product of the expected size (approximate 1.4 kb) was digested with the
I restriction enzymes and inserted into the
-marked plasmid pRS315 (ATCC77144, 6.02 kb) to construct plasmid pME1376 (7.3 kb).
was transformed by the lithium acetate method
cells carrying these plasmids were grown in synthetic complete medium lacking tryptophan (SC-TRP
) or leucine (SC-LEU
YPH499 transformants harboring plasmid pRS315 (vector control) or pME1376 (pRS315-
) were grown in selective medium overnight and diluted to an OD
of 0.2 with sterile SC-LEU
medium. Afterwards, 5-fold serial dilutions were prepared, and 20 µl of each was spotted on the SC-TRP plate.
Primers for degenerate PCR were designed on the basis of a multiple alignment of published yeast
. A PCR product with an approximate size of 0.4 kb was amplified, TA-cloned, and sequenced. The alignment of the deduced amino acid sequence encoded by the amplified fragment with those of Trp1 homologs suggested that the cloned fragment contains a conserved
sequence, which was named
. On the basis of the sequenced gene fragment, primers for genome walking were designed to isolate the 5’- and 3’-untranslated regions of the
gene (data not shown). The entire nucleotide sequence of
, including 300 bp of the 5’ and 3’ regions, was obtained by genome walking and sequence assembly, and deposited in the GenBank under Accession No. KR078268.
Analysis of the
upstream sequence revealed two TATA-like sequences
at nucleotide positions -200 and -53. Potential transcription initiation sites
were found, which had consensus motifs TC(G/A)A (at nucleotide positions -90 and -97) or RRYRR (a pyrimidine surrounded by two purines; positions -136 and -270). A possible CAAT box
was found at nucleotide position -135.
gene consists of an ORF of 759 bp and encodes a putative 252-amino-acid polypeptide with an estimated molecular mass of 27.6 kDa, which is larger than the 24.1 kDa protein in
. The calculated pI was 5.7, similar to 5.34 in
. Sequence analysis indicated that the
gene does not contain introns. Sequence alignment with other Trp1 proteins (
) indicated that
Trp1 has a typical primary structure; the previously reported Trp1 domains
are highly conserved in
Trp1 protein shares the highest sequence identity (43.5%) with that of
, followed by those of
(38.4%). The alignment also confirmed several motifs highly conserved among fungal Trp1s: VGVFRNOS at amino acid positions 99–105, DFVQLHG at 120–126, and ILAGGLT at 200–206 (
. Analysis by the C-DART program confirmed that
Trp1 shares a conserved structure with the PRAI family
Alignment of multiple amino acid sequences from S. fibuligera Trp1 and other homologous yeast Trp1. Fully conserved residues are marked in black. The conserved regions on which degenerate primers were designed are marked with asterisks and hydrophobic conserved regions are in boxes. The numbers on the left and right indicate the positions of the amino acids. Saccharomycopsis fibuligera (GenBank Accession No. KR078268); Candida albicans (XP_718951); Candida glycerinogenes (ABU53939); Candida orthopsilosis (EAZ63723); Komagataella pastoris (CAA04452); Saccharomyces cerevisiae (CAA24634); Scheffersomyces stipitis (EAZ63723); Wickerhamomyces anomalus (AAO19636).
The ability of
to complement the
phenotype was tested by transformation of the
strain YPH499. The cloned
gene with its own promoter and terminator was inserted into the low-copy plasmid pRS315. As shown in
S. cerevisiae trp1
strain harboring pRS315 (vector control) was unable to grow on a SC-TRP
plate. However, the
transformant harboring the plasmid pME1376 grew successfully on SC-TRP - medium. These results indicated that the
Complementation of S. cerevisiae trp1mutation by SfTRP1. S. cerevisiae YPH499 strain expressed SfTRP1 from plasmid pME1376. Cultures were grown to mid-log phase in SC-LEU - medium and diluted to an OD600 of 0.2 with the same medium. Then, 5-fold serial dilutions were prepared, and each dilution was spotted onto the plate. Cells were incubated at 30℃ and photographed after 3 days.
In conclusion, we successfully isolated the full-length
gene through degenerate PCR and genome walking. The functionality of the cloned
gene was confirmed by complementation of
auxotrophy in a
strain. Thus, a new marker gene to engineer starch-utilizing yeast
is now available.
This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009993),” Rural Development Administration, Republic of Korea.
The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences.
DOI : 10.1002/elps.11501401163
Aromatic amino acid biosynthesis in the yeastSaccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.
Isolation and characterization of theTRP1gene from the yeastYarrowia lipolyticaand multiple gene disruption using a TRP blaster.
DOI : 10.1002/yea.987
Characterization of starch-utilizing yeastSaccharomycopsis fibuligeraisolated from Nuruk.
Korean J. Microbiol. Biotechnol.
DOI : 10.4014/kjmb.1409.09006
Multiple alignment using hidden Markov models.
Proc. Int. Conf. Intell. Syst. Mol. Biol.
Haissam Jijakli M
Cloning and sequence analysis of theTRP1gene encoding the phosphoribosyl anthranilate isomerase fromPichia anomala(strain K).
DOI : 10.1002/yea.1033
CDART: protein homology by domain architecture.
DOI : 10.1101/gr.278202
Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
de Figueroa LIC
Optimized amylolytic enzymes production inSaccharomycopsis fibuligeraDSM-70554: an approach to efficient cassava starch utilization.
Enzyme Microb. Technol.
DOI : 10.1016/j.enzmictec.2007.10.005
Fermentation of cellobiose to ethanol by industrialSaccharomycesstrains carrying the β-glucosidase gene (BGL1) fromSaccharomycopsis fibuligera.
DOI : 10.1016/j.biortech.2011.01.062
1H and 15N NMR investigation of the interaction of pyrimidine nucleotides with ribonuclease A.
Eur. J. Biochem.
DOI : 10.1111/j.1432-1033.1985.tb08708.x
Multiple control elements in theTRP1promoter ofSaccharomyces cerevisiae.
Mol. Cell Biol.
Cloning of theCandida glabrata TRP1andHIS3genes, and construction of their disruptant strains by sequential integrative transformation.
DOI : 10.1016/0378-1119(95)00552-H
Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast.
DOI : 10.1007/s00294-007-0142-1
Nucleotide sequences ofSaccharomycopsis fibuligeragenes for extracellular beta-glucosidases as expressed inSaccharomyces cerevisiae.
Appl. Environ. Microbiol.
Roles of the aromatic residues conserved in the active center ofSaccharomycopsisalphaamylase for transglycosylation and hydrolysis activity.
DOI : 10.1021/bi00168a009
Transformation of lithium-treated yeast cells and the selection of auxotrophic and dominant markers.
Methods Mol. Biol.
Cloning and disruption of thePichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6genes and their use as auxotrophic markers.
DOI : 10.1002/yea.1202
Characterization of theCandida albicans TRP1gene and construction of a homozygoustrp1mutant by sequential co-transformation.
DOI : 10.1016/0378-1119(94)90687-4
Cloning and characterization of the orotidine-5'-phosphate decarboxylase gene (URA3) from the osmotolerant yeastCandida magnoliae.
J. Microbiol. Biotechnol.
DOI : 10.4014/jmb.1111.11071
Saccharification of cassava starch bySaccharomycopsis fibuligeraYCY1 isolated from Loog-Pang (rice cake starter).
Songklanakarin J. Sci. Technol.
Cold Spring Harbor Laboratory Press
Molecular characterization ofTRP1, a gene coding for tryptophan synthetase in the basidiomyceteCoprinus cinereus.
DOI : 10.1016/0378-1119(89)90338-7
Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant ofSaccharomycopsis fibuligera.
J. Ind. Microbiol. Biotechnol.
DOI : 10.1007/s10295-011-0943-6