Poster apresentado em XXI International Conference on Yeast Genetics and Molecular Biology, Gotemburgo, Suécia, 7 Jul. - 12 Jul. 200

Casal, Margarida

Ferreira, Pedro Moradas

Paiva, Sandra

Queirós, Odília

Universidade do Minho: RepositoriUM

Molecular and physiological characterization of mono- and dicarboxylic acids permeases in the yeast Kluyveromyces lactis

O. Queirós 1,2,3, S. Paiva 1, P. Moradas-Ferreira3,4and M. Casal11Departamento de Biologia da Universidade do Minho, 2Instituto Superior de Ciências da Saúde-Norte, Paredes 3Instituto de Biologia Molecular e Celular, Universidade do Porto, 4Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto.RESULTS AND DISCUSSIONK. lactis is able to use mono- and dicarboxylic acids as the sole carbon and energy source and presents mediated transport systems for these acids. S.cerevisiae although presents a carrier for monocarboxylates, is not able to transport dicarboxylic acids.The gene encoding lactic acid permease has been cloned in S. cerevisiae (JEN1 gene). Using the Génolevures database, two clones of K. lactis (named KlJEN1 and KlJEN2) containing a partial sequence of two genes homologues to JEN1 were identified. The RACE technique allowed the identification of the whole sequence of these genes. KlJEN1 and KlJEN2 expression has been assayed, and although in S. cerevisiae JEN1 expression is glucose repressed, a transcript of both JEN1/2 genes is present in glucose grown cells of K. lactis, indicating a different regulation in this species. Indeed, the analysis of the KlJEN1/2promoters sequenced so far did not reveal cis elements for repressors like mig1p, in opposition to what happens with ScJEN1 promoter.On the second part of this work, a K. lactis gene, associated with the regulation of malate utilization (MUP1) is presented. This gene was isolated by heterologous gene expression in S. cerevisiae. A second copy of this gene is present in the K. lactis genome (MUP2) and functional analysis of MUP1 and MUP2was carried out by the construction of a K. lactisΔ mupΔ1mup2 deleted strain. The growth in YNMal is very similar in the three strains; however, concerning dicarboxylic acid transport, the double mutant presented half of the activity for this transport system.  INTRODUCTIONThe physiological and biochemical characterisation of permeases for carboxylic acids has been described in the literature for several yeast species. In the present work we focused on the genetic and molecular studies carried out in the mono- and dicarboxylate transport systems in Kluyveromyces lactis:Previous studies developed in the species S. cerevisiae led to the identification of the lactate permease gene (JEN1/YKL217w) by our group. In Part I we describe the cloning and in silico analysis of 2 homologues of JEN1 found in the non-conventional yeast K. lactis (KlJEN1 and KlJEN2). Results concerning lactic acid transport by this species are presented as well as some results concerning expression of KlJEN1 gene,associated with the carbon source used in the media.K. lactis is able to use malic acid as the sole carbon and energy source. In the Part II of this work we describe the identification of a K. lactisgene, associated with the regulation of malate utilization (MUP1), which was detected by heterologous gene expression in S. cerevisiae. The medium YPmalate with the pH indicator bromocresol purple (YPMI medium) was used for the screening of the transformants. Southern analysis revealed the presence of a second copy of this gene in the K. lactis genome (MUP2) and functional analysis of MUP1 and MUP2 was carried out by the construction of a K. lactisΔ mupΔ1mup2 deleted strain.I – Cloning and in silico analysis of KlJEN1/2II – JEN1/2 expression in different carbon sourcesMolecular and physiological characterization of mono- and dicarboxylic acids permeases in the yeast Kluyveromyces lactisV – Cloning of MUP1, a gene involved in malate utilisationIV – Dicarboxylic acid transport in K. lactis5101520253035- 20 0 20 40 60 80 100 1201/[malic acid] (mM)1/V (nmol. s-1. mg-1d.w.)-1Initial uptake rates of labelled acid as a function of its concentration in K. lactis malic acid grown cells:A) Uptake of malic acid  in the absence of other dicarboxylic acids;  in the presence of succinic acidB) Uptake of succinic acidVI – Characterization of mup1Δ mup2ΔK. lactis double mutant0,11100 10 20 30O.D. 60000,20,40,60,81,21,40 0,2 0,4 0,6 0,8 1,0 1,21,0Succinic acid (mM)V (nmol. s-1. mg-1d.w) – K. lactis MUP1 MUP2S – K. lactis mup1Δ MUP2 – K. lactis mup1Δ mup2Δz – K. lactis MUP1 MUP2{ – K. lactis mup1Δ MUP2 – K. lactis mup1Δ mup2ΔGrowth in YNMal Succinic acid transportSearch in GénolevuresDatabaseIdentification of 2 K. lactis clonescontaining JEN1 homologues partial sequences (KlJEN1 and KlJEN2)Identification of the whole sequences of KlJEN1 and KlJEN2 by RACEKlJEN1 cloningRegion identified in the Génolevures database5’RACEKMM1KlJEN13’RACERegion identified in the Génolevures database5’RACE200 pbKMM1KlJEN23’RACEKlJEN2 cloningTransmembrane domains in KlJEN1 and KlJEN2KlJEN2KlJEN1Probable localization of Kljen1p and Kljen2p in the plasma membrane(PSORT software analysis)III – Lactic acid transport in K. lactisJEN1/2 expression:-Levels of JEN1/2 transcript after 4 h incubation in YNB containing the indicatedcarbon source:   (-) absent;  (+) present; (ND) not determinedRecovery of the plasmid:pO1 containing MUP1 geneWT - wild type;T(pO1)-pO1 transformant.T(pO1)WTGenomic libraryof K. lactisUra+uraS. cerevisiaeIdentificationPhysiological characterisationGrowth in YPmalate medium and malic acid consumption:WT: J, OD600nm; z, L-malic acid concentration;T(pO1): J,OD600nm;z,L-malic acid concentration.Time (h)0 10 20 30 40 50 60 70[L-malic acid] (% w/v)5.0z0.10.20.30.40.50.60.70.8  0.010.11.0 z zzzzz z z0zV (nmol.s-1.mg-1dw)OD600nmSequencingPartial restriction map and DNA sequencing strategy of the insert contained in pO1. MUP1 gene was found to be responsible for the phenotype observed and it is duplicated in the K. lactis genomeSequencing reactionsDraISalI HindIIIEcoRI PvuIIHpaI ScaI HindIIIEcoRVBglII1 kbSacIXbaIEcoRIORF1 ORF2 = MUP1396 bp 1062 bpuraura ura uraStrainglucose glycerolJEN1 + + + ++S. cerevisiaeW303 (JEN1)++ NDCarbon sourcemalicacidcitricacidethanol+ ++ +ND ++ -lacticacidaceticacid--1/[succininc acid] (mM)-11/V(nmols-1.mg-1d.w.)-10123456780 20 40 60 80 100 120A BKINETIC  PARAMETERS:Malic acid: Km = 0.12 mM malic acid and Vmax = 0.47 nmol malic acid s-1 mg-1 d.w.Succininc acid:Km = 0.04 mM succinic acid and Vmax = 0.75 nmol succinic acid s-1 mg-1 d.w.JEN2 + + + + + ++ +K. lactisYP pyruvic acid-grown cellsKINETIC  PARAMETERS, pH 5.0:Km = 4.3 mM lactic acid acidVmax = 1.97 nmol s-1 mg-1 d.w.0 1 2 3 4 5 6 7 80.00.51.01.5[DL-lactic acid] (mM)V(nmols-1mg-1dry wt.)

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Molecular and physiological characterization of mono- and dicarboxylic acids permeases in the yeast Kluyveromyces lactis

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Universidade do Minho: RepositoriUM