Isolation of the Phosphoribosyl Anthranilate Isomerase Gene (TRP1) from Starch-Utilizing Yeast Saccharomycopsis fibuligera
Isolation of the Phosphoribosyl Anthranilate Isomerase Gene (TRP1) from Starch-Utilizing Yeast Saccharomycopsis fibuligera
Journal of Microbiology and Biotechnology. 2015. Sep, 25(8): 1324-1327
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : May 11, 2015
  • Accepted : May 14, 2015
  • Published : September 28, 2015
Export by style
Cited by
About the Authors
Eun-Hee Park
Myoung-Dong Kim

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera 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 S. fibuligera was 43.5% homologous to that of Komagataella pastoris . The cloned TRP1 gene ( SfTRP1 ) complemented the trp1 mutation in Saccharomyces cerevisiae , suggesting that it encodes a functional TRP1 in S. fibuligera . A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.
Gene transformation or disruption would be a useful tool to analyze the molecular mechanisms in yeast [16 , 21] . Genetic transformation systems employing auxotrophic markers, such as URA3, TRP1, HIS3, and LEU2 , have been developed for different yeasts, because transformants can be easily selected on drop-out media [19 , 23] . The TRP1 gene encodes phosphoribosyl anthranilate isomerase, which catalyzes the third step in tryptophan biosynthesis [2] and is commonly used as a selectable marker in yeasts [6 , 8 , 15] .
Saccharomyces fibuligera is the major amylolytic yeast found in starchy substrates, such as bread dough, alcoholic beverages, and rice cake starters [5 , 24] . Co-cultures of S. fibuligera with Saccharomyces cerevisiae , Candida utilis , or Zymomonas mobilis are used to produce ethanol from starch [4 , 11] . These co-cultures produce amylase, protease, β-glucosidase, and trehalose, and have applications in food, biofuel, and pharmaceutical industries [12 , 17 , 18 , 27] . In our previous study, a thermotolerant S. fibuligera strain was isolated from nuruk , a traditional Korean starter for rice wine fermentation [5] .
In this study, we isolated and sequenced the TRP1 gene from S. fibuligera 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 SfTRP1 was demonstrated by complementation of a TRP1 - negative S. cerevisiae strain.
S. fibuligera [5] and S. cerevisiae YPH499 [ MAT a ura3-52 lys2-801_amber ade2-101_ochre trp1-∆63 his3-∆020 leu2-∆1 ] (Clontech, Palo Alto, CA, USA) were used. Escherichia coli 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, S. fibuligera 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 [3 , 6] . 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. E. coli cells were transformed as described previously [25] . 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 ( ) [7] . 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.
To obtain SfTRP1 with its own promoter and terminator regions, approximately 300 bp of 5’ and 3’ regions of the SfTRP1 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 Sac I and Sal I restriction enzymes and inserted into the LEU2 -marked plasmid pRS315 (ATCC77144, 6.02 kb) to construct plasmid pME1376 (7.3 kb). S. cerevisiae was transformed by the lithium acetate method [10] . S. cerevisiae cells carrying these plasmids were grown in synthetic complete medium lacking tryptophan (SC-TRP - ) or leucine (SC-LEU - ).
The S. cerevisiae YPH499 transformants harboring plasmid pRS315 (vector control) or pME1376 (pRS315- SfTRP1 ) were grown in selective medium overnight and diluted to an OD 600 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 TRP1 gene sequences [3 , 6 , 8 , 22] . 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 TRP1 sequence, which was named SfTRP1 . On the basis of the sequenced gene fragment, primers for genome walking were designed to isolate the 5’- and 3’-untranslated regions of the SfTRP1 gene (data not shown). The entire nucleotide sequence of SfTRP1 , 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 SfTRP1 upstream sequence revealed two TATA-like sequences [14] at nucleotide positions -200 and -53. Potential transcription initiation sites [13] 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 [26] was found at nucleotide position -135.
The SfTRP1 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 S. cerevisiae [14] . The calculated pI was 5.7, similar to 5.34 in S. cerevisiae [1] . Sequence analysis indicated that the SfTRP1 gene does not contain introns. Sequence alignment with other Trp1 proteins ( Fig. 1 ) indicated that S. fibuligera Trp1 has a typical primary structure; the previously reported Trp1 domains [8 , 20] are highly conserved in S. fibuligera Tp1. S. fibuligera Trp1 protein shares the highest sequence identity (43.5%) with that of Komagataella pastoris , followed by those of Candida albicans (39.5%), C. orthopsilosis (39.1%), and Wickerhamomyces anomalus (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 ( S. fibuligera numbering) [3 , 8 , 22] . Analysis by the C-DART program confirmed that S. fibuligera Trp1 shares a conserved structure with the PRAI family [9] .
PPT Slide
Lager Image
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 SfTRP1 to complement the trp1 phenotype was tested by transformation of the S. cerevisiae strain YPH499. The cloned SfTRP1 gene with its own promoter and terminator was inserted into the low-copy plasmid pRS315. As shown in Fig. 2 , the S. cerevisiae trp1 strain harboring pRS315 (vector control) was unable to grow on a SC-TRP - plate. However, the S. cerevisiae transformant harboring the plasmid pME1376 grew successfully on SC-TRP - medium. These results indicated that the TRP1 gene from S. fibuligera complements the trp1 mutation in S. cerevisiae .
PPT Slide
Lager Image
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 SfTRP1 gene through degenerate PCR and genome walking. The functionality of the cloned TRP1 gene was confirmed by complementation of trp1 auxotrophy in a S. cerevisiae strain. Thus, a new marker gene to engineer starch-utilizing yeast S. fibuligera 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.
Bjellqvist B , Hughes GJ , Pasquali C , Paquet N , Ravier F , Sanchez JC 1993 The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14 1023 - 1031    DOI : 10.1002/elps.11501401163
Braus GH 1991 Aromatic amino acid biosynthesis in the yeastSaccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway. Microbiol. Rev. 55 349 - 370
Cheon SA , Han EJ , Kang HA , Ogrydziak DM , Kim JY 2003 Isolation and characterization of theTRP1gene from the yeastYarrowia lipolyticaand multiple gene disruption using a TRP blaster. Yeast 20 677 - 685    DOI : 10.1002/yea.987
Chi Z , Chi Z , Liu G , Wang F , Ju L , Zhang T 2009 Saccharomycopsis fibuligeraand its applications in biotechnology. Biotechnol. Adv. 27 423 - 431    DOI : 10.1016/j.biotechadv.2009.03.003
Choi DH , Park EH , Kin MD 2014 Characterization of starch-utilizing yeastSaccharomycopsis fibuligeraisolated from Nuruk. Korean J. Microbiol. Biotechnol. 42 407 - 412    DOI : 10.4014/kjmb.1409.09006
Cosano I , Alvarez P , Molina M , Nombela C 1998 Cloning and sequence analysis of thePichia pastoris TRP1, IPP1andHIS3genes. Yeast 14 861 - 867    DOI : 10.1002/(SICI)1097-0061(19980630)14:9<861::AID-YEA276>3.0.CO;2-N
Eddy SR 1995 Multiple alignment using hidden Markov models. Proc. Int. Conf. Intell. Syst. Mol. Biol. 3 114 - 120
Friel D , Vandenbol M , Haissam Jijakli M 2003 Cloning and sequence analysis of theTRP1gene encoding the phosphoribosyl anthranilate isomerase fromPichia anomala(strain K). Yeast 20 1331 - 1337    DOI : 10.1002/yea.1033
Geer LY , Domrachev M , Lipman DJ , Bryant SH 2002 CDART: protein homology by domain architecture. Genome Res. 12 1619 - 1623    DOI : 10.1101/gr.278202
Gietz RD , Woods RA 2002 Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350 87 - 96
González CF , Fariña JI , de Figueroa LIC 2008 Optimized amylolytic enzymes production inSaccharomycopsis fibuligeraDSM-70554: an approach to efficient cassava starch utilization. Enzyme Microb. Technol. 42 272 - 277    DOI : 10.1016/j.enzmictec.2007.10.005
Gurgu L , Lafraya A , Polaina J , Marin-Navarro J 2011 Fermentation of cellobiose to ethanol by industrialSaccharomycesstrains carrying the β-glucosidase gene (BGL1) fromSaccharomycopsis fibuligera. Bioresour. Technol. 102 5229 - 5236    DOI : 10.1016/j.biortech.2011.01.062
Hahn U , Desai-Hahn R , Ruterjans H 1985 1H and 15N NMR investigation of the interaction of pyrimidine nucleotides with ribonuclease A. Eur. J. Biochem. 146 705 - 712    DOI : 10.1111/j.1432-1033.1985.tb08708.x
Kim S , Mellor J , Kingsman AJ , Kingsman SM 1986 Multiple control elements in theTRP1promoter ofSaccharomyces cerevisiae. Mol. Cell Biol. 6 4251 - 4258
Kitada K , Yamaguchi E , Arisawa M 1995 Cloning of theCandida glabrata TRP1andHIS3genes, and construction of their disruptant strains by sequential integrative transformation. Gene 165 203 - 206    DOI : 10.1016/0378-1119(95)00552-H
Ma Y , Sugiura R , Saito M , Koike A , Sio SO , Fujita Y 2007 Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast. Curr. Genet. 52 97 - 105    DOI : 10.1007/s00294-007-0142-1
Machida M , Ohtsuki I , Fukui S , Yamashita I 1988 Nucleotide sequences ofSaccharomycopsis fibuligeragenes for extracellular beta-glucosidases as expressed inSaccharomyces cerevisiae. Appl. Environ. Microbiol. 54 3147 - 3155
Matsui I , Yoneda S , Ishikawa K , Miyairi S , Fukui S , Umeyama H , Honda K 1994 Roles of the aromatic residues conserved in the active center ofSaccharomycopsisalphaamylase for transglycosylation and hydrolysis activity. Biochemistry 33 451 - 458    DOI : 10.1021/bi00168a009
Mount RC , Jordan BE , Hadfield C 1996 Transformation of lithium-treated yeast cells and the selection of auxotrophic and dominant markers. Methods Mol. Biol. 53 139 - 145
Nakai R , Sen K , Kurosawa S , Shibai H 2000 Cloning and sequencing analysis of Trp1 gene ofFlammulina velutipes. FEMS Microbiol. Lett. 190 51 - 56    DOI : 10.1111/j.1574-6968.2000.tb09261.x
Nett JH , Hodel N , Rausch S , Wildt S 2005 Cloning and disruption of thePichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6genes and their use as auxotrophic markers. Yeast 22 295 - 304    DOI : 10.1002/yea.1202
Ostrander DB , Gorman JA 1994 Characterization of theCandida albicans TRP1gene and construction of a homozygoustrp1mutant by sequential co-transformation. Gene 148 179 - 185    DOI : 10.1016/0378-1119(94)90687-4
Park EH , Seo JH , Kim MD 2012 Cloning and characterization of the orotidine-5'-phosphate decarboxylase gene (URA3) from the osmotolerant yeastCandida magnoliae. J. Microbiol. Biotechnol. 22 642 - 648    DOI : 10.4014/jmb.1111.11071
Saelim K , Dissara Y , Kittikun AH 2008 Saccharification of cassava starch bySaccharomycopsis fibuligeraYCY1 isolated from Loog-Pang (rice cake starter). Songklanakarin J. Sci. Technol. 30 65 - 71
Sambrook J , Russell DW 2011 Molecular Cloning. Cold Spring Harbor Laboratory Press New York
Skrzynia C , Binninger DM , Alspaugh JA , Pukkila PJ 1989 Molecular characterization ofTRP1, a gene coding for tryptophan synthetase in the basidiomyceteCoprinus cinereus. Gene 81 73 - 82    DOI : 10.1016/0378-1119(89)90338-7
Wang DS , Zhao SF , Zhao MX , Li J , Chi ZM 2011 Trehalose accumulation from cassava starch and release by a highly thermosensitive and permeable mutant ofSaccharomycopsis fibuligera. J. Ind. Microbiol. Biotechnol. 38 1545 - 1552    DOI : 10.1007/s10295-011-0943-6