Yeast 15, 1393–1398 (1999)

The URA5 Gene Encoding Orotate-phosphoribosylTransferase of the Yeast Kluyveromyces lactis: Cloning,Sequencing and Use as a Selectable Marker

XIYUAN BAI1†, MICHAEL LARSEN1 AND FRIEDHELM MEINHARDT1*1Westfalische Wilhelms-Universitat Munster, Institut fur Mikrobiologie, Corrensstrasse 3, D-48149 Munster,Germany

A pair of degenerate primers was used for amplification and cloning of an internal fragment of the K. lactis URA5gene. Primers were designed on the basis of highly conserved motifs within protein sequences predicted for URA5genes from several microorganisms. Using the amplified fragment as a probe, we finally cloned and sequenced a1·9 kb chromosomal fragment containing the orotate-phosphoribosyltransferase-encoding URA5 gene and anincomplete open reading frame strikingly similiar to SEC65 of Saccharomyces cerevisiae and other yeasts, in whichthe gene encodes a subunit of the signal recognition particle. Uracil-requiring mutants of K. lactis CBS 683 wereselected on media containing 5-fluoro-orotic acid and used as recipients in transformation experiments using K. lactisURA5 as the selectable marker, thereby proving functionality of the cloned gene. The sequence presented herehas been submitted to the EMBL data library under Accession No. AJ001358. Copyright ? 1999 John Wiley &Sons, Ltd.

— Kluyveromyces; orotate-phosphoribosyltransferase; URA5; SEC65

*Correspondence to: F. Meinhardt, Institut fur Mikrobiologie,Universitat Munster, Corrensstrasse 3, D-48149 Munster,Germany. Tel: +49-251-83-39825; fax: +49-251-83-38388;e-mail: meinhar@uni-muenster.de†Present address: Xi’an Jiaotong University, School of Chemi-cal Engineering, Institute of Bioscience and Biotechnology,West Xiangning Road 28, Xian, China 710049.Contract/grant sponsor: State Education Commission of the

INTRODUCTION

Kluyveromyces lactis is among the most importantindustrial yeast species, able to grow abundantlyon inexpensive media, such as lactose and whey.As a generally regarded as safe (GRAS) organism,it is ideally suited for use in food industries. Theorganism has long been used for large-scale pro-duction of lactase and single cell protein(Bonekamp and Oosterom, 1994). Expression of

People’s Republic of China (Beijing).

CCC 0749–503X/99/131398–06$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

heterologous genes was achieved by applyinggenetic engineering techniques (Romanos et al.,1992); its high protein secretion capability makesK. lactis superior even to Saccharomyces cerevisiae.Human interleukin-1â has been expressed ef-ficiently in K. lactis; secretion of this protein wasfound to be 80–100-fold higher than in baker’syeast (Fleer et al., 1991a); bovine prochymosin andhuman serum albumin were expressed similarly(van den Berg et al., 1990; Fleer et al., 1991b).

Genetic engineering requires stable and non-reverting strains as recipients in transformationexperiments. Wild-type strains can only be trans-formed using dominant selectable markers, such asthe bacterial aminogylcoside phosphotransferasegene, conferring resistance to aminoglycoside anti-biotics (Sreekrishna et al., 1984; Tanguy-Rougeau

et al., 1990). However, selection systems based on

Received 22 March 1999Accepted 26 April 1999

1394 X. BAI ET AL.

Figure 1. Cloning of the Kluyveromyces lactis URA5 gene. (A) Primers used for amplification ofan internal URA5 fragment were designed according to the given protein sequences (see also Figure3). (B) Southern blot analysis using the amplified fragment as a probe. Lane 1, Bulk DNA of S.cerevisiae AH22, digested with EcoRI; lane 2, K. lactis bulk DNA digested with HindIII, EcoRI(lane 3), BamHI (lane 4) and PstI (lane 5). (C) Maps of plasmids pBK2·6 and pBK4·5 containingfragments of the K. lactis URA5 gene, and pBKLUR5 containing the entire URA5 coding regionand an incomplete open reading frame homologous to S. cerevisiae SEC65. The HindIII–EcoRIfragment of pBKLUR5 is 1·9 kb in size and corresponds to the sequence outlined in Figure 2.

those marker genes are of minor significance inapplied microbiology because of the generalproblems brought about by antibiotics in large-scale fermentations. In this contribution, wedescribe the isolation and sequencing of the K.lactis URA5 gene and transformation experimentswith the gene as a selectable marker.

The ability to synthesize pyrimidines is commonto pro- and eukaryotic organisms, however,the pathway has undergone a number of changesand modifications during evolution (for review,see Jones, 1980). The last two steps in thebiosynthesis of pyrimidines are catalysed byorotate-phosphoribosyl-transferase (OPRTase, EC2.4.2.10) and orotidine-5*-phosphate decarboxy-lase (OMPDase, EC4.1.1.23), respectively. Inmammals and slime moulds (Dictyostelium discoi-deum), both enzymatic activities are combined on a

Copyright ? 1999 John Wiley & Sons, Ltd.

single polypeptide chain and thus are encoded as abifunctional enzyme (Boy-Marcotte et al., 1984).In bacteria such as Escherichia coli and Salmonellatyphimurium (O’Donovan and Neuhard, 1970), infilamentous fungi such as Neurospora crassa(Caroline, 1969), and in the yeast Saccharomycescerevisiae (Jund and Lacroute, 1972) both enzy-matic activities are encoded by separate genes. Thenucleotide sequence of the URA3 gene of Kluy-veromyces lactis encoding the OMPDase is alreadyknown (Shuster et al., 1987). We searched for theURA5 gene of this yeast species and determined itssequence, which eventually allowed us to establishthe primary structure of the OPRTase and tocompare it with enzymes from organisms pre-viously described. Furthermore, it offered the abil-ity to use the gene as a genetic marker in cloningand transformation experiments.

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1395URA5 GENE OF K. LACTIS

MATERIALS AND METHODS

Yeast strains K. lactis CBS683 and S. cerevisiaeAH22 (MATa, leu2-3, 3-112, his4-519, can1,[rho0]) were grown on YEPD (1% yeast extract, 2%peptone, 2% glucose) or minimal medium (0·67%yeast nitrogen base, 2% glucose) supplementedwith uracil (50 mg/l). Selection of fluoro-oroticacid-resistant mutants was carried out at 1 g/l.Escherichia coli strain JM107 (F* traD36 lacIq

Ä(lacZ)M15 proA+B+/e14"(McrA") Ä(lac-proAB) thi gyrA96 (Nalr) endA1 hsdR17(r"

K m+K)

relA1 supE44) was used in transformation exper-iments and for amplification of nucleic acids(Yanisch-Perron et al., 1985).

Standard cloning procedures were carried out asdescribed previously (Sambrook et al., 1989).Chromosomal DNA of yeasts was isolated accord-ing to Sherman et al. (1986). For DNA hybrid-ization experiments, the DIG-dUTP system(Boehringer–Mannheim, Germany) was usedaccording to recommendations given by the manu-facturer. Transformation of yeasts was performedusing the LiAc method (Gietz and Schiestl, 1995).PCR for amplification of a 131 bp URA5 genefragment was carried out in a thermocycler(Minicycler=, MJ Research Inc., USA) accordingto the following protocol. After the first denatur-ation step (95)C, 5 min), 35 cycles were run: 94)C,1 min; 60)C, 0·5 min; 72)C, 1 min. For DNAsequencing the SequiTherm EXCEL= II Long-Read= DNA Sequencing Kit-LC (Epicentre Tech-nologies, Wisconsin, USA), with IRD41-labelledoligonucleotides (MWG-BIOTECH, Germany)was used; electrophoresis and nucleic acid detec-tion was done on a LI-COR> 4000L automaticsequencer (Li-COR Inc., Nebraska, USA).

RESULTS AND DISCUSSION

Copyright ? 1999 John Wiley & Sons, Ltd.

Figure 2. Sequence of a 1·9 kb chromosomal K. lactis frag-ment containing the URA5 gene and a part of a S. cerevisiaeSEC65 homologue. Predicted amino acid sequences are givenunderneath using the one letter code.

Isolation and sequencing of the URA5 geneBy aligning known protein sequences deduced

from URA5 genes of several microorganisms, i.e.bacteria, yeasts and filamentous fungi, we ident-ified conserved regions within the OPRTasepolypeptide sequence. Based on these data, a pairof degenerate primers was synthesized that allowedPCR amplification of a 131 bp fragment usingK. lactis bulk DNA as the template (see Materialand Methods and Figures 1A and 2). Theamplified DNA was cloned in E. coli and itssequence determined. When the nucleotide se-quence was compared to the corresponding part of

the S. cerevisiae URA5 gene, 78% identity becameevident. The cloned fragment was labelled with

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Figure 3. Alignment of OPRTases of several microorganisms. Conserved amino acids arehighlighted. Source of sequences: EMBL data library. Kluyveromyces lactis (Accession No.O13474), Saccharomyces cerevisiae URA5 (P13298), Saccharomyces cerevisiae URA10 (P30402),Yarrowia lipolytica (P41923), Colletotrichum graminicola (P35788), Metarhizium anisopliae(O42767), Podospora anserina (P08309), Sordaria macrospora (P18904), Trichoderma reesei(P21846), Escherichia coli (P00495), Haemophilus influenzae (P43885), Pseudomonas aeruginosa(P50587)

Copyright ? 1999 John Wiley & Sons, Ltd.

Functional analysis of the K. lactis URA5 geneCompairing the deduced amino-acid sequence

of the putative K. lactis URA5 gene to otherknown sequences demonstrated that the encoded

polypeptide is indeed very similar to proteins

DIG-dUTP and used as a probe in Southernanalysis of genomic K. lactis DNA digested withdifferent restriction endonucleases. The results ofthese hybridization experiments are presented inFigure 1B.

Two of the hybridizing fragments, i.e. the 4·5 kbHindIII fragment and the 2·6 kb PstI fragment,were excised from the gel, eluted and subsequentlycloned in E. coli using pUCBM20 (Boehringer–Mannheim, Germany) as the vector, eventuallyresulting in plasmids pBK4·5 and pBK2·6, respect-ively. Candidate clones were identified by colonyfilter hybridization with the labelled 131 bp frag-ment as the probe. Plasmids were isolated fromhybridizing clones and restriction enzyme mapsconstructed. Both cloned inserts were found to bepartially colinear (Figure 1C).

For determination of the URA5 sequence, wesubcloned a 1·29 kb EcoRI/HindIII fragment ofpBK4·6 and a 0·99 kb PstI/HindIII fragment ofpBK2·6. Both fragments are indicated as whitebars in Figure 1C; they overlap one another in the0·3 kb PstI/HindIII internal region. By applyingstandard cloning procedures, we constructed plas-mid pBKLUR5 (Figure 1C), which is a pUCBM20

derivative, with a 1·3 kb insert carrying the entireURA5 gene of K. lactis CBS 683. The insertwas sequenced and assigned EMBL databaseAccession No. AJ001358. In Figure 2 the predictedpolypeptide of the URA5 gene is given, along withthe nucleotide sequence. Downstream of theURA5 gene we identified an additional inverselyorientated open reading frame with striking simi-larities (82%) to SEC65 of S. cerevisiae andother yeasts, which encodes a subunit of thesignal recognition particle (Stirling and Hewitt,1992). The genetic organization as such appearsto be highly conserved among yeasts, becauselocalization of SEC65 downstream of URA5 isalso reported for Candida albicans (Regnacqet al., 1998), S. cerevisiae (Stirling and Hewitt,1992) and Yarrowia lipolytica (Sanchez et al.,1995, 1997).

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1397URA5 GENE OF K. LACTIS

identified as OPRTases from other sources(Figure 3).

Functionality of the URA5 gene was furtherinvestigated in transformation experiments usinguracil-requiring mutants of strain K. lactis CBS683 as recipients and the 1·9 kb EcoRI/HindIIIfragment cloned in pUCBM20 as the transformingagent (see Figure 1C, plasmid pBKLUR5).Uracil-requiring mutants were obtained using5-fluoro-orotic-acid (FOA) as the selecting com-pound. Clones resistant to FOA are affected ineither OMPDase or OPRTase (Boeke et al., 1984)and are thus uracil-requiring auxotrophicmutants. Randomly chosen FOA-resistant cloneswere checked for their ability to grow on minimalmedium with and without supplementation ofuracil (data not shown). Eight randomly chosenuracil-requiring mutants were subsequentlyused as recipients in transformation experimentsusing pBKLUR5 DNA (Figure 1C). In S. cerevi-siae, loss of URA5 function can be complementedpartly by the URA10 gene product, whichrepresents an OPRTase isoenzyme (see also Figure3), resulting eventually in a leaky phenotypeof S. cerevisiae ura5 mutants (de Montigny et al.,1990). Since no leaky mutants were obtained, aURA10 homologue is unlikely to be presentin K. lactis CBS683. Surprisingly, all of the eightrandomly chosen uracil-requiring, FOA-resistantmutants could be transformed to prototrophyonly with K. lactis URA5 (integrated into vectorpBKLUR5). Consistent with these finding isthe fact that no transformants were obtainedwhen the S. cerevisiae URA3 gene was used intransformation experiments (data not shown).Thus, FOA-resistant mutants of K. lactis CBS683were found to be predominantly affected in URA5:the reason for this phenomenon remains at presentobscure. The fact that all mutants tested were hitin URA5 might, however, be indicative for URA3duplication in strain CBS683.

For this approach to isolate genes it is centraland necessary to deduce primers from highly con-served motifs and adapt them to the codon usageof the organism under investigation, as exemplifiedfor URA5 of K. lactis. The developed methodmight, thus, potentially apply to clone URA5 genesfrom other microorganisms.

ACKNOWLEDGEMENTS

We thank B. Zonnefeld (Department of Cell

Biology and Genetics, University of Leiden–

Copyright ? 1999 John Wiley & Sons, Ltd.

Cluisius Laboratory, The Netherlands) for provid-ing strain K. lactis CBS683. X. Y. Bai wassupported by a grant from the State EducationCommission of the People’s Republic of China(Beijing).

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