Kluyveromyces marxianus Vectors 347

Construction of Efficient Centromeric, Multicopy andExpression Vectors for the Yeast Kluyveromycesmarxianus Using Homologous Elements and thePromoter of a Purine-Cytosine-Like Permease

Received July 14, 1999; revised September 16, 1999; accepted Septem-ber 30, 1999. *For correspondence. Email iborra@igmors.u-psud.fr;Tel. +33-1-69 15 46 75; Fax. +33-1-69 15 72 96.

J. Mol. Microbiol. Biotechnol. (1999) 1(2): 347-353.

© 1999 Horizon Scientific Press

JMMB Research Article

María M. Ball1, Alain Raynal2, Michel Guérineau2,and François Iborra2*

1Present address: Laboratorio Organizacion y Expresióndel Gen, Dpto. de Biología, Facultad de Ciencas, Mérida5101 Venezuela2Laboratoire de Biologie et Génétique Moléculaire,Institut de Génétique et Microbiologie, U.M.R. C.N.R.S.8621, Bât 400 Université Paris 11, 91 405 Orsay CedexFrance

Abstract

Efficient centromeric and multicopy vectors have beenconstructed for the yeast Kluyveromyces marxianususing homologous ARS and centromere sequences.A homologous promoter of a purine–cytosine per-mease gene called PCPL3 has been cloned, using anexpression system based on GUS. Its strength hasbeen estimated in K. marxianus by putting the homolo-gous β-glucosidase gene under its control. This pro-moter is very efficient as activities higher than the onesobtained with the Saccharomyces cerevisiae PGK pro-moter were obtained. This promoter appears to beconstitutive in various conditions tested. Its five tran-scription start sites have been mapped, and a deriva-tive expression vector for K. marxianus has been con-structed.

Introduction

Kluyveromyces marxianus is a yeast closely related to K.lactis. Many studies have shown that these yeasts are re-lated to Saccharomyces cerevisiae (Lachance, 1993). Inthese three species, many genes have been character-ized and have been shown to be often interspecifically func-tional and similarly organised (Webster and Dickson, 1988)and regulated by similar effectors (Breunig and Kruger,1987). In some cases regulatory genes have been swappedfrom one yeast to another and proved to exert the samecontrol (Oberye et al., 1993). Very often, similar UAS orURS are found in their promoters, and some are subjectto similar regulations (Ruzzi et al., 1987; Cassart et al.,1997). Nevertheless, K. marxianus is more suitable thanS. cerevisiae for biological purposes. K. marxianus is usedindustrially for the production of biomass by growth on whey

and production of alcohol by growth on inuline rich plants(Bajpai and Bajpai, 1991; Brady et al., 1994). It is also asource of β−galactosidase and inulinase, two enzymesabsent in S. cerevisiae. Inulinase can be secreted up to1g/l, a yield by far much higher than the one obtained forthe similar enzyme in S. cerevisiae. This yeast also hasthe property of growing at high temperatures and is con-sequently easier to handle in industrial fermentation.

Such “non conventional yeasts” whose secretion isvery efficient are consequently used increasingly to pro-duce metabolites and heterologous proteins difficult orimpossible to obtain in S. cerevisiae. For a review see Fleeret al. (1992). As classic genetics is poorly developed inmost of these yeasts, one has to develop efficient andappropriate vectors. From both evolutionary and biotech-nology points of view, it was interesting to make this yeastaccessible to genetic engineering. So far, the only vectoravailable was a poorly efficient replicative vector using anARS from the yeast K. lactis constructed with a selectionfor G418 (Das and Hollenberg, 1982). It was then adaptedfor a uracil selection (Iborra, 1993). Using this vector, ura3mutants were isolated and a high efficiency transforma-tion method was developed (Iborra, 1993). ARS sequencesand two centromeres of K. marxianus have been clonedand described. They function in K. lactis but not in S.cerevisiae (Iborra and Ball, 1994). Using these elements,it was possible to construct homologous efficient vectorsincluding expression vectors. In this context, it was inter-esting to isolate new promoters from K. marxianus andanalyse their expression in this yeast assuming that ho-mologous promoters will allow a better expression of thegenes placed under their control. The cloning of these pro-moters was undertaken using a fusion with the β−glucuro-nidase (GUS) reporter gene.

In this paper, we report the construction of homolo-gous K. marxianus multicopy and centromeric vectors aswell as the cloning of a very active promoter used to cons-truct an efficient expression vector. This promoter controlsthe expression of a gene, PCPL3, similar to one memberof the purine-cytosine permease family of S. cerevisiae.The accession number for the described sequence isAJ011419.

Results and Discussion

Construction of Transforming Vectors forK. marxianus (Figure 1A)Multicopy Plamid Vector pKM2(Escherichia coli-K. marxianus)The 810 bp sequence of fragment B, KpnI-ClaI previouslyisolated from the genomic DNA of K. marxianus (Iborraand Ball, 1994) contains the origin of replication KMARS2.

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348 Ball et al.

This fragment was inserted in the AatII site of pRS306 af-ter rendering all ends blunt by treatment with the nucleaseS1 (Figure 1A). This plasmid is much more stable than theones based on the ARS of K. lactis. Its loss rate is only12% per generation.

Centromeric Plasmid Vector pKMCA (E. coli-K. marxianus)The 800 bp ClaI-SacI sequence from fragment A, previ-ously isolated from the genomic DNA of K. marxianus, con-tains the origin of replication KMARS1 and the centromereA. First, the inner HindIII site was suppressed after diges-tion with HindIII. Then, this fragment was inserted in theAatII site of pRS306 after rendering all ends blunt by treat-ment with the Klenow enzyme. This plasmid has a lossrate of 3% per generation. As the ARS and centromeres ofK. marxianus function in K. lactis, but not in S. cerevisiae(Iborra and Ball, 1994), these vectors can be used in K.lactis, but not in S. cerevisiae.

Promoter Cloning with the GUS Reporter SystemConstruction of pYEL1/GUS Plasmid (Figure 1B)Since K. marxianus has a strong endogenous β−galac-tosidase activity, another reporter gene was used: the GUSgene reporter system. It has been successfully used toclone plant promoters, but rarely for yeasts. β−glucuroni-dase activity is as easy to detect as β−galactosidaseusing X-glc and pNPG. The SalI-SstI fragment containingthe modified GUS gene was inserted in the XhoI-BglII sitesof plasmid pYEL1, a S. cerevisiae-E. coli shuttle plasmid.This places the GUS gene between the S. cerevisiae PGKpromoter and its terminator, two well characterised andhighly efficient sequences used to express heterologousproteins in S. cerevisiae.

Construction of the Promoter CloningVector pKM2/GUS (Figure 1C)The 2.2 kb BamHI-SalI fragment of pYEL1/GUS contain-ing the GUS gene and the PGK terminator was inserted inthe BamHI-SalI sites of pKM2. This leads to a promoterlessGUS gene followed by the PGK terminator.

Construction of the Library2 to 5 kb Sau3A fragments resulting from a partial diges-tion of K. marxianus genomic DNA were cloned in theBamHI site upstream of the GUS coding sequence in theplasmid pKM2/GUS (Figure 1C). Thus, sequences havinga promoter activity can be detected by the expression ofthe GUS gene. After transforming yeast cells, 6000 URA+

clones were examined and about 100 positive clones weredetected qualitatively by their blue colour afterpermeabilisation in presence of X-glc and quantitativelyby the yellow coloration using pNP-GUS as a substrateafter lysing the cells with zymolyase. The GUS activity wasspectrophotometrically measured for the most stronglycoloured clones.

Cloning the PromoterAlthough, 6000 clones are not expected to contain thewhole genome of K. marxianus, it was sufficient to checkthe feasibility of the method and to find an efficient pro-moter. Clone 3 was chosen because it gave the highestlevel of GUS expression among the 10 most active clones.The insert of the clone 3 plasmid was sequenced. It re-vealed an ORF of 103 bp amino-acids fused in phase withthe β−glucuronidase gene and preceded by a promoterlike sequence.

Figure 1. Vector ConstructionA, construction of a replicative(pKM2) or a centromeric (pKMCA)shuttle vector E. coli - K. marx-ianus. DNA fragments containingeither the origin of replicationKMARS2 or the origin of replica-tion KMARS1 + Centromere Awere inserted in the AatII site ofpRS306 after rendered bluntended. MCS = multiple cloningsites. Β, cloning of the β−glucuro-nidase gene between the pro-moter and the terminator of the S.cerevisiae PGK gene: the SalI-SstI fragment of plasmid pBI101was inserted between the XhoIand BglII sites of pYEL1 after be-ing rendered blunt. C, constructionof the GUS reporter vector ofpKM2: fragment SalI-BamHI of thepYEL1-GUS plasmid was insertedbetween the SalI-BamHI sites ofpKM2. D, expression vectorpKM2-β−glu: Chosen promoter isinserted using the added XhoI sitein the polylinker of PKM2 plasmidbetween XhoI and another con-venient site among the remainingsites. Then, the β−glucosidasegene plus its terminator on a SalI-SalI fragment was introduced inthe XhoI site at the end of the pro-moter.

Kluyveromyces marxianus Vectors 349

Characterization of the PCPL3 GeneIt was of interest to know the nature of the gene under thecontrol of the clone 3 promoter. Since the initial 103 amino-acids of the gene product were not sufficiently informative,the gene was cloned and sequenced and complete cod-ing sequence was compared to sequence databanks.

A K. marxianus DNA library constructed in the plasmidYEP352 was screened, using a SpeI-XhoI fragment of thepromoter as a probe. One clone strongly hybridised withthe probe. It contained a plasmid with a 10.5 kb insert. Thepromoter and the gene were localised after restrictionmapping and hybridisation with the probe. DNA fragmentscontaining the gene were then sequenced (Figure 2).Analysis of the DNA sequence revealed an ORF of 1686bp coding for a putative 562 amino-acid protein with apredicted molecular weight of 63.17 kDa. Comparisons withsequences databanks of this predicted protein showedsequence identities with S. cerevisiae genes which had

been classified in the purine/cytosine permease family, asubfamily of the major facilitator super family (MFS)characterised by two structural units of six transmembranespanning α−helical segments connected by a cytoplasmicloop (Nelisen et al., 1997). In S. cerevisiae, four genesconstitute the purine/cytosine permease family: the purine/cytosine permease gene FCY2 (Weber et al., 1990), twogenes FCY21 and FCY22 located as tandem copies onchromosome V, and a YGL186C ORF located onchromosome VII with lower identity.

The best scores were obtained with the YGL186C ORF(Coglievina et al., 1977) of unknown function coding for anhypothetical protein of 64.5 kDa. The gene product was63% identical in a 587 amino acid overlap with YGL186Cand 25% identical to FCY2 (Figure 3). Blast and Beautyanalysis (Worley et al., 1995) indicates three conserveddomains between these three proteins. The gene wasnamed PCPL3 for Purine-Cytosine Family n° 3.

Figure 2. Sequence of thePCPL3 Gene and of its Pro-moterPutative TATA boxes areboxed. The T rich sequence isheavily underlined. STRE se-quences are in italics and un-derlined: AGGGG. IncompleteGCN4 sequences are under-lined: TGACTN

350 Ball et al.

Figure 3. Comparison of the Deduced Protein Sequences of the S. cerevisiae Purine Cytosine Permease Gene Family and of the K. marxianus PCPL3 GeneOn first line, Sc1 is the S.cerevisiae FCY2 protein . On the second line, Sc2 is the S. cerevisiae YGL186C protein and on the third line Km is the K.marxianusPCPL3 protein. Black shaded amino acids are conserved in the three proteins, grey shaded amino acids are conserved between two proteins.

Kluyveromyces marxianus Vectors 351

Analysis of the PCPL3 PromoterStructure of the PromoterThe sequence of the PCPL3 promoter (Figure 2) containstypical promoter elements. They are very similar to the onesof S. cerevisiae genes: TATA, TATAAA and TATATA se-quences, as well as a T-rich sequence found in variouspromoters between the TATA box and the initiation site(Heslot and Gaillardin, 1992). Five RNA initiation sites werefound by the primer extension method before the putativeTATA boxes: four major ones at positions -55, -96, -100,-104 and a weaker one at-109 (Figure 4).

Functional AnalysisIn order to analyse the expression of this promoter in K.marxianus a XhoI site was introduced by PCR upstreamof the ATG to remove the coding sequence and a BamHIsite at - 1049 bp to facilitate cloning (Figure 5). As a re-porter gene, the endogenous β−glucosidase gene of K.marxianus was used rather than β-glucuronidase. It is wellexpressed in S. cerevisiae (Raynal and Guérineau, 1984)and was found to give more reliable results. The low en-

dogenous activity in K. marxianus did not impair activitymeasurements. The modified promoter was then insertedeither in pKM2 to get multicopy plasmids or in pKMCA toget centromeric plasmids. A SalI-SalI fragment containingthe β−glucosidase ORF followed by its terminator wasligated in the XhoI site (Figure 1D).

Potential Regulatory SequencesThree STRE consensus sequences (AGGGG) (Marchleret al., 1993) were found in this promoter (Figure 2). Theyare responsible upon stress conditions of the transcrip-tional induction of several S. cerevisiae genes. This se-quence is present in the promoter of Hsp26 gene. To in-vestigate their functionality, the yeast cells containing therecombinant gene proPCPL3-β−glucosidase-terPGK in thecentromeric vector pKMCA were submitted to variousstresses. Clearly in K. marxianus, these sequences bythemselves are not sufficient to confer any stress inducedregulation of the gene, since all the stress conditions tested(heat shock, oxidative shock, saline shock) did not inducea higher expression.

Six incomplete GCN4 consensus target sequences(TGACTN) were also found in this promoter. Although thiscore sequence has been proved to be essential to dere-press upon amino acids starvation the synthesis of mostamino acids, we examined the possibility of a generalamino-acid regulation for the expression of this gene in K.marxianus, since the exact target of such regulation is notknown in this yeast and could be slightly different. There isonly a two fold effect upon addition of amino-acids both inK. marxianus and in S. cerevisiae, which may be explainedby the more rapid growth of the cells in medium supple-mented with all amino acids.

The expression of the fused gene is not sensitive tocatabolic repression. Only a slight increase is observedwhen the cells are shifted from 2% glycerol to 2% glucose,although there is a two fold increase when cells are grownon 4% glucose. When nucleotides such as guanine, cyto-sine or adenine are added to the medium, no effect on theproduction of β-glucosidase have been noticed. Therefore,the PCPL3 gene appears to be constitutive for the variousconditions tested.

To check the efficiency of the promoter PCPL3,promoters of Sacharomyces cerevisiae (GAL10, PGK,HSP26) and ADH1 of K. marxianus were modified in the

Figure 4. Transcription Start SiteInitiation starts of transcription for the promoter PCPL3 in K. marxianusdetermined by primer extension (-55, -96, -100, -104).

Figure 5. PCR ModificationModification brought by PCR to promoters and β−glucosidase used in thisstudy. Modifications are indicated in bold small letters.

5' PCPL 3 3'TTGATCTAGTTTCC ACAGAATAATATGGgATCc cTcgagBamHI XhoI

HSP70 TCATATTCCAACAATG ADHI CATACACAACACATG ctCgAg CtcgaG

PGK CAAATTAACATG HSP26 TAAAAACAATG cTcgAg ctcgaG

GAL1-10 GAAAATTCAATATAAATG CtcgAg

β-glucosidase gtcgaCCtcgACGTGCAAACGTAAGAAAAAAAATGTCTAAATTTGA SalI

Hsp26

Hsp70

352 Ball et al.

same way (Figure 5). A XhoI site was introduced 5' of theATG (Figure 2), then inserted in pKM2 and a SalI-SalIfragment containing the ORF of the β−glucosidase geneand its terminator was ligated in this new XhoI site. As acontrol, the same construction was used with the PGKpromoter of S. cerevisiae.

After transformation of K. marxianus, the transformantswere grown to early stationary phase in selective mediumand the β−glucosidase activity was determined in cell ex-tracts. Several clones for each construction were checked.Figure 6 shows that the activity of the PCPL3 promoter isabout the same as the one obtained with the PGK pro-moter in K. marxianus. It is also worth noticing that all S.cerevisiae inducible promoters which were tested were alsoinducible in K. marxianus but with an induction level some-what lower (Johnston, 1987; Bossier et al., 1989). The highactivity of the PCPL3 promoter is not due to a titration ef-fect of some repressor by a multicopy vector as high levelof expression were also obtained by a centromeric one.

Deletion AnalysisIn order to further analyse this promoter two deletions wereconstructed and analysed. When the promoter is short-ened to the HindIII site at -256, there is a 25% increase ofpromoter activity in K. marxianus (Figure 6). Further re-duction to -200 resulted in a loss of activity, although thepromoter still contains the TATA box and the five initiationsites found by primer extension. Only an incomplete GCN4core consensus binding sequence can be found in the -256-200 fragment. No other known regulatory sites are presentin this fragment. This region contains two repeats of poly Tfollowed by poly A sequences. It is possible that they couldform a secondary structure important for the transcriptionof an unknown site of regulation. The reason why a per-mease promoter is so active in K. marxianus is unknownand would be worth further investigation.

Construction of an Expression VectorThe high activity obtained with the -256 promoter deletionled to the construction of a small sized expression vectorwith a better expression than the one obtained with thePGK promoter. Several restriction sites of the polylinkerare available in front of the promoter and can be used toinsert regulatory sequences which might be very usefulfor specific needs. This vector can be either multicopy usingpKM2 or centromeric using pKMCA as starting vectors.The new vectors for the yeast K. marxianus, containinghomologous ARS and centromere sequences appear tobe very efficient. The replicative vector is more stable thanthe only other vector available constructed with an ARSfrom K. lactis. Despite the high strength of the promoter inthe expression vector, no instability of the vectors carryingit was observed. With the cloned PCPL3 promoter, and its–256 bp derivative, an efficient expression vector is nowavailable for this yeast.

Experimental procedures

Strains and Growth MediaK. marxianus FI 47 ura3 was derived by UV mutagenesis from ATCC 12424strain, a homothallic strain. It was grown on YPD rich medium or YNΒ−glucose (Difco) minimal medium supplemented by necessary auxotrophicrequirements. It was transformed by electroporation using a Jouan squarepulse generator (Iborra, 1993), or by the Li-PEG method of Ito et al. (1983).E. coli strain DH5α (BRL) was propagated on LB medium. It was renderedcompetent and transformed by the method of Inoue et al. (1990).

PlasmidspRS306, a Bluescript KS (Stratagene) derivative containing the S. cerevisiaeURA3 gene without a yeast origin of replication was obtained from DrSikorski (Sikorski and Hieter, 1989). Plasmid pYEL1 and pYEL3 were con-structed by Nicaud et al. (1994). Plasmid pBI101 and GUS kit were ob-tained from Clontech. They were prepared by the method of Berghammerand Auer (1993) or by alkaline lysis. Plasmid YEP352 was constructed byHill et al.(1986).

Colony Detection and Enzymes ActivitiesA replica of yeast colonies was made on a Whatman filter paper W540.The filter was frozen at -70°C, then thawed. It was then deposited on asolution of 67 mM phosphate buffer pH7 containing zymolyase and 5-bromo-4-chloro-3 indolyl-glucuronide (X-glc) and incubated at 30°C. The deepestblue colonies were selected. Cells extracts were obtained after lysis bytreatment with zymolyase or disruption with glass beads followed by a 15mn centrifugation in a microfuge. Protein concentration was measured onthese extracts by the method of Bradford (1976). Then, the hydrolysis ofparanitrophenyl(pNP)Glucuronide and pNPGlucoside by the cell extractswas followed by the increase of absorbancy at 425 nm in 67mM phosphatebuffer pH7 with an EM of 13 106 to measure the activity of the β−glucuroni-dase. The same conditions were used for assaying the β−glucosidase ac-tivity. Specific activity is expressed in nmoles of pNPG hydrolyzed per mgof protein per minute.

The following conditions were used to induce the expression of pro-moters:ADHIGrowth on selective medium + glucose 5%. The cells were then washedand put in a selective medium + 2% ethanol during 6 hours (Ladrière et al.,1993; Ladrière, 1995).GALIGrowth on selective medium + 0.2% raffinose, 0.2% glucose. The cellswere then washed and put in a selective medium + 2% galactose during24h.Hsp26Growth on selective medium up to stationary phase.Stress Conditions UsedCells transformed by the centromeric vector pKMCA containing the β−glu-cosidase gene under the control of the PCPL3 promoter were grown onYPD up to DO 0.5. They were then transferred in YPD medium containingeither NaCl 0.3 M, or Sorbitol 0.4 M, or H2O2 and gathered after one or twohours of induction. Thermic shock was produced by shifting the cells grownat 28 to 50°C for K. marxianus. Such high temperatures were used for K.marxianus since optimal growth temperature for this strain is 37°C.General Amino Acids RepressionThe cells were grown on minimal medium, then washed and grown in thesame medium with 0.2% casaminoacids.

Figure 6. β−Glucosidase Production in K. marxianus under the Control ofVarious Yeasts PromotersThe β−glucosidase activity was measured in constructions where the β−glucosidase gene is under the control of various promoter of K. marxianusand S. cerevisiae. The conditions of induction were described in materialand methods. -1000, -256 and -200 refer to the length of PCPL3 promoter.

PGK

S. c.

ADH

1 K.

m.

GAL

1-10

S. c

.

HSP

26 S

. c.

PCPL

3 K.

m. -

1000

PCPL

3 K.

m. -

256

PCPL

3 K.

m. -

200

promoter

non inducedinduced

0

5000

10000

activity

Kluyveromyces marxianus Vectors 353

Polymerase Chain Reaction (PCR)PCR DNA amplification were done on a TECHNE thermocycler (OSI) us-ing Thermus acquaticus DNA polymerase from Eurogentec or Appligene.

Primer ExtensionPrimer extension was performed with mRNA isolated with Hybond-mAPpaper (Amersham) using the primer extension kit of Promega.

Double Strand DNA SequencingIt was performed on both strands on Exonuclease III nested deletions(Pharmacia kits) or on restriction fragments using the dideoxy terminationmethod, either manually or on AB1373 Stretch sequencer (PE AppliedBiosystems) using a Dye Terminator kit. DNA sequences were comparedto the EMBL library using the BLASTP + BEAUTY programs (Altschul etal., 1990; Worley et al. 1995). Protein sequences were compared using theMultialign version 5.3.3. of Corpet (1988).

Acknowledgements

The authors gratefully acknowledge Dr. Bolotin-Fukuhara and Dr. Carl Mannfor critical reading of the manuscript.

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