JouRNAL OF BACrERIOLOGY, Feb. 1994, p. 596-6010021-9193/94/$04.00+0Copyright X) 1994, American Society for Microbiology

Lactobacillus plantarum ldhL Gene:Overexpression and Deletion

THIERRY FERAIN, DOMINIQUE GARMYN, NATHALIE BERNARD,PASCAL HOLS, ANi JEAN DELCOUR*

Laboratoire de Gene'tique Moleculaire, Unite de Gnetique, Universite Catholique de Louvain,B-1348 Louvain-la-Neuve, Belgium

Received 1 July 1993/Accepted 30 November 1993

Lactobaciflsplantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-laCtate withstereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene

(designated idhL) that encodes L-(+)-lactate dehydrogenase from L. plustanm DG301 was cloned bycomplementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of320 aminoacids closely related to that of LactobaciUlus pentosus. A multicopy plasmid bearing the ldhL gene withoutmodification of its expression signals was introduced in L. plantarum. L-LDH actity was increased up to13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)-and D-(-)-lactate. A stable chroiosomal deletion in the ldhL gene was then constructed in L. plantarum by a

two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activityand in exclusive production of the D isomer of lactate. However, the global concentration of lactate in theculture supernatant remained unchanged.

Lactobacillus plantarum is a lactic acid bacterium that con-verts glucose to pyruvate via the Embden-Meyerhof-Parnaspathway. Most of the pyruvate is reduced to lactate, which isthe major fermentation end product. However, less efficientways of dissipating pyruvate such as pyruvate formate-lyase(20) and acetolactate synthase (28) pathways have also beendescribed for L. plantarum.

Lactate dehydrogenases (LDH) are responsible for the finalconversion of pyruvate to lactate with the concurrent oxidationof NADH formed during glycolysis. Some lactic acid bacteria,includingL. plantarum, produce both L-(+)- and D-(- )-lactatefrom pyruvate by distinct and stereospecific enzymes calledL-LDH (EC 1.1.1.27) and D-LDH (EC 1.1.1.28), respectively.In these species, the ratio of L-(+)- to D-(-)-lactate secretedinto the culture supernatant changes with growth stage andconditions (14). Other lactic acid bacteria have only one LDHand therefore produce only one isomer of lactate. Althoughthe type of lactate formed is used in the classification of lacticacid bacteria (35), the physiological role of each enzyme andtheir corresponding products have not yet been investigated.Indeed, in addition to being a metabolic end product, lactate isalso involved in several energetic processes. In the absence ofglucose, L. plantarum is able to use lactate as an energy source.This metabolic ability has been described under both aerobicand anaerobic conditions (20, 29) and seems to involve distinctpathways. Lactate is also involved in the energy recyclingprocess which generates metabolic energy by end-productefflux (19, 37). The extrusion of protons in symport with lactatevia specific carriers is a source of energy because it contributesto the generation and the maintenance of a proton motiveforce across the cytoplasmic membrane. This proton motiveforce is an essential driving force in metabolic processes suchas amino acid transport (11). The importance of lactate in

* Corresponding author. Mailing address: Departement de Biologie,Unite de Genetique, Universite Catholique de Louvain, BatimentClaude Bernard, Place Croix du Sud, 5 bte 6, 1348 Louvain-la-Neuve,Belgium. Phone: 32 (10) 473484. Fax: 32 (10) 473109. Electronic mailaddress: delcour@gene.ucl.ac.be.

these physiological and metabolic roles has been established insome lactic acid bacteria, but the possible advantage of pro-ducing and extruding one or both isomers has not yet beeninvestigated.

In this work, we have metabolically engineered L. plantarumso as to alter its L-LDH activity. To this end, we have clonedand sequenced the ldhL gene and used it to construct strainseither overexpressing or not expressing L-LDH. These strainswere then used to investigate the possibility and the conse-quences of modifying the ratio of D-(-)- to L-(+)-lactate inthis bacterium.

MATERUILS AND METHODS

General molecular biology techniques were essentially per-formed according to the instructions given by Sambrook et al.(32).

Bacterial strains, plasmids, and media. The bacterial strainsand plasmids used in this study are listed in Table 1. L.plantarum was grown anaerobically in MRS broth (Difco 0881)at 37C without shaking. For complementation assay, Esche-richia coli FMJ144 was grown anaerobically at 37°C on M9minimal medium plates supplemented with 0.4% glucose and0.2% Casamino Acids. Antibiotics were used at the followingconcentrations: erythromycin, 250 ,ug/ml for E. coli and 5

,ug/ml for L. plantarum, and chloramphenicol, 50 p.g/ml for E.coli and 10 ,ug/ml for L. plantarum. Plasmids pLAB1301 (18)and pJDC9 (5) have been described elsewhere.DNA purification and cotistruction of a genomic library.

Plasmid DNA was isolated from L. plantarum according to themethod of Posno et al. (30), and chromosomal DNA wasisolated from crude cell lysates as previously described (3).Chromosomal DNA from L. plantarum DG301 was subjectedto partialAlul digestion. The 1- to 7-kbp fragments isolated byagarose gel electrophoresis were cloned into the SmaI site ofplasmid pJDC9.

Transformation. Electrotransformation of E. coli and L.plantarum was performed as described by Dower et al. (10) andJosson et al. (18), respectively.

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ldhL OF L. PLANTARUM 597

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Characteristic(s)a Source or reference

StrainsL. plantarumDG301 Wild-type strain TEXEL (Dange-Saint-

Romain, France)NCIB8826 Wild-type strain LMG (Ghent, Belgium)TF101 NCIB8826 AldhL This study

E. coliFMJ144 Aldh pfl::Camr trpA trpR his-29(Am) pro-2 arg-427 deoB arc tsx IN(rrnD-rrnE) lacY 23TG1 suoE hsdA5 thiA(lac-proAB)F' (traD36) ProAB+ lacIq lacZAM15 15

PlasmidspJDC9 Emr; AlacZ 5pLAB1301 Emr Cmr; shuttle vector E. coli-L. plantarum 18pGIT005 Emr; pJDC9 with a 1.65-kbp AluI fragment from L. plantarum DG301 This studypGIT032 Emr Cmr; pLAB1301 with a 1.45-kbp KpnI fragment from pGIT005 containing IdhL This studypGIP711 Cmr Apr; shuttle, unstable integration vector containing the origin of replication from 17

plasmid pE194pGIT015 Emr; same as pGIT005 with a 305-bp deletion (HpaI-NruI fragment) inside the open This study

reading frame of the IdhL genepGIT017 Emr; same as pGIT015 with a XbaI-PstI fragment from pGIP711 containing the origin This study

of replication from plasmid pE194b

a Emr, Apr, and Cmr indicate resistance to erythromycin, ampicillin, and chloramphenicol, respectively.b See reference 38.

PCR amplification of DNA. L. plantarum chromosomalDNA was amplified by PCR with 1 to 5 ,ug of DNA in a finalvolume of 100 ,ul containing deoxyribonucleoside triphos-phates (200 ,uM each), oligonucleotides (25 ,uM each), 50 ,uMtetramethylammonium chloride, and 2.5 U of Taq DNApolymerase (Boehringer Mannheim) and the buffer suppliedwith the enzyme. Amplification was performed as follows, witha LEP Scientific (PREMIII) heating block: cycle 1, 99°C for 5min, 40°C for 1 min, and 72°C for 2 min; cycles 2 to 29, 92°Cfor 1 min, 42°C for 1 min, and 72°C for 2 min; and cycle 30,92°C for 1 min, 42°C for 1 min, and 72°C for 10 min.Single-stranded DNA for sequencing reactions was synthesizedby asymmetric PCR adapted from the work of McCabe (25).DNA sequencing. Sequencing reactions were performed by

the primer walking strategy with the Sequenase kit (U.S.Biochemical) on double-stranded plasmid DNA or on single-stranded DNA from asymmetric PCR (25).LDH and lactate assays. LDH activity was measured on

crude cell extracts. L. plantarum cells from 10-ml culturesgrown anaerobically were harvested by centrifugation, resus-pended in a final volume of 1 ml of phosphate buffer (pH 5.6)(KH2PO4 [73 mM], Na2HPO4 [3.5 mM]), and broken mechan-ically with a Braun homogenizer (three times during 30 s withan equal volume of 0.18-mm glass beads). Cellular fragmentswere eliminated by centrifugation at 12,000 x g. The LDHassay was performed as previously described (3). ResidualL-LDH activity corresponds to the activity measured aftercrude cell extracts are heated for 3 min at 50°C to inactivateD-LDH enzyme (27). Total protein in cell extracts was mea-sured according to the method of Lowry et al. (22). One unit ofactivity corresponds to the oxidation of 1 ,umol of NADH permin.

Concentrations of L-(+)- and D-(-)-lactate in culture su-pernatants were measured enzymatically with BoehringerMannheim kit no. 1112821.

Nucleotide sequence accession number. The ldhL nucleotidesequence reported in this paper will appear in EMBL, Gen-Bank, and DDBJ data bases under accession number X70926.

RESULTS AND DISCUSSION

Cloning of the ldhL gene of L. plantarum in E. coli. The ldhLgene from L. plantarum DG301 was cloned by complementa-tion of the E. coli FMJ144 strain deficient for lactate dehydro-genase and pyruvate formate-lyase (23). Previous works haveshown that anaerobic growth of this strain can be restored bycomplementation with an ldh gene (3, 9). A genomic libraryfrom L. plantarum DG301 was prepared in plasmid pJDC9,and about 45,000 recombinant plasmids were transferred intoE. coli FMJ144 for complementation. Twenty-six recombinantcandidates restoring anaerobic growth of the mutant host wereisolated. LDH activity was detected in crude cell extracts of all26 clones, which were shown to harbor overlapping inserts bySouthern hybridization experiments (data not shown). PlasmidpGIT005, expressing the highest L-LDH level, was chosen forfurther experiments. The genomic origin of the pGIT005 insertwas confirmed by Southern blot analysis of L. plantarumDG301 chromosomal DNA (data not shown).DNA sequence of the ldhL gene. The complete nucleotide

sequence of the pGIT005 insert is shown in Fig. 1. It spans1,651 bp and contains an open reading frame of 320 codonsstarting with a TTG, which has already been reported as aninitiation codon for some genes of gram-positive bacteria (26).A putative ribosome binding site complementary to the 3' endof L. plantarum 16S rRNA (EMBL accession number M58827)is present at canonical distance, with a calculated free energyof duplex formation (AGf) of -18 kcal/mol (Fig. 1, boldunderline). Several putative promoters ( - 35 and - 10 boxes)have also been observed in the 5' noncoding region (Fig. 1,double underline). Inspection of the 3' noncoding sequenceshows a putative transcriptional termination signal consistingof an inverted repeat sequence followed by a cluster of T thatcould form a stem and loop structure in mRNA with acalculated free energy of -12.6 kcal/mol (Fig. 1, horizontalarrows).

Analysis of the amino acid sequence revealed typical fea-tures of the well-characterized L-LDH family (for a recentcomparative analysis, see reference 21). According to the

VOL. 176, 1994

598 FERAIN ETAL.J.BcROL

AC.00T.A.CC.CC0.TTCGO.A. .T. .GCGTC0.CCA. .CA0..G.0....GCAC. .. .ACACCA.TTTAG.TG.GGC0.C.C................ G.

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A.G.TGOA.C.TGA.TGOT.A G.0GTTGT.OOGCG. .AATTTOTCA.GAATCATOAT.A.AGC.TGCCAOTTGG. .0G.-T . .C.G.TCO.CAOCCO. .0G. .CGCCTT-AG.CCGACC.CCOG 1262

,GOAOO,A,C,C-AACTT,A,ATT-A,TGA-AA,C'AT-A-AACO'TOGA-CT-TA'TOATTOOOGOCAOTATCCTrCOOTCTrCCTCOTCATCGGATATOTTGCCACGAO 1051

FIG. 1. Nucleotide sequence of the L. plantarum DG301 ldhL gene and deduced amino acid sequence. Two putative transcriptional promoters(-35 and -10 boxes) are double underlined. A putative ribosome binding site (RBS) which exhibits complementarity to the sequence3'-CUUUCCUCC-5' at the 3' end of the L. plantarum 168 rRNA (EMBL accession number M58827) is bold underlined. A likely terminatorstructure is indicated by horizontal arrows. Comparisons with the corresponding idhiL gene and L-LDH protein of the closely related species L.pentosus (ATCC 8041, erroneously named L. plantarum in the work of Taguchi and Ohta [36]) are also illustrated; nucleotide substitutions arenoted above the L. plantarum nucleotide sequence, and the five corresponding amino acid substitutions are indicated below the amino acidsequence.

description and numbering of Eventoff et al. (12), the NADH-

binding site GxGxxG(17x)D common to all NAD-dependent

dehydrogenases starts at position 28. This site comprises an

aspartate (D-53) which is known to discriminate between NAD

and NADP (13). The two amino acids involved in acid-base

catalysis (H-1-95 and D-168) and a series of amino acids

involved in substrate binding and specificity (R-171, Q-102,

T-246, R-109) can also be identified by comparison (6, 7).

Among other bacterial L-LDH, the highest degree of identitywas observed with that of the closely related species Lactoba-

cilluspentosus (36). Comparison of the two genes reveals 91%

identity between nucleic acid sequences within the open read-

ing frames. Most of these substitutions are conservative,

resulting in more than 98% identity in amino acid sequences.

This high degree of similarity begins about 200 bp upstream of

the IFIG initiation codon and extends up to the stop codon.

These observations are in agreement with the fact that L.

pentosus was considered synonymous with L. piantarum until a

few years ago (39). By contrast, nucleic acid sequences are

extremely divergent in the 5' and 3' flanking regions. These

observations confirm more recent taxonomy studies which

show that L. piantarum and L. pentosus are closely related but

nevertheless distinct species (39).

Overexpression and deletion of the IdhL gene in L. planta-

rum. The id/iL gene was reintroduced on a multicopy plasmidin L. plantarum with the objective of increasing L-LDH activity

through gene dosage effect. We wanted to analyse the conse-

quences of this perturbation on the global production of

lactate and on the ratio Of L to D isomers. The id/iL gene was

inserted in a shuttle vector, pLABl3Ol (18). The resulting

plasmid, called pGIT032, was introduced into L. plantarum

NCIB8826 by electrotransformation. Recombinant and controlstrains were grown anaerobically for 30 h in selective MRSbroth, and total LDH activity was measured in crude cellextracts. Residual activity was also measured in crude cellextracts after heat treatment (3 min at 500C), which is knownto inactivate D-LDH in L. plantarum (27). These data (Table 2)show a large increase in specifiC L-LDH activity associated withthe multicopy plasmid (13-fold increase) compared with thecontrol vector. The impact of increased L-LDH activity onlactate production was then investigated by measuring concen-trations Of L-(+)- and D-( -)-lactate in culture supernatants.Data listed in Table 2 show that the drastic increase in L-LDHactivity observed above had hardly any effect on the productionOf L-(+)- and D-( -)-lactate. Additional measurements per-formed at various culture times in the growth and the station-ary phases show that the L-LDH activity is largely increasedduring the whole culture, while the D-( -)- to L-(+)-lactateratio remains virtually unchanged. These observations per-formed on the overexpressing strain demonstrate that lactateproduction is not limited by LDH activity. Similarly, otherstudies describing overexpression of glycolytic and fermenta-tive enzymes in E. coli (2, 34), Zymomonas mobilis (1), andSaccharomyces cerevisiae (4, 8, 33) reported small effects onglucose flux.The next attempt to perturb the lactic acid fermentation

pathway was the construction of an L. plantarum id/L-deficientstrain. The procedure used leads to a completely stablechromosomal deletion within the idhiL gene through a two-stephomologous recombination process (Fig. 2). In short, thedeletion is first constructed in vitro on an intermediate vector.This plasmid bears a copy of the idhiL gene (Ll/L3) lacking an

J. BAcrERIOL.

ldhL OF L. PLANTARUM 599

TABLE 2. LDH activity and lactate production in L. plantarum as a function of IdhL gene dosage and in strain deleted for this gene

L. plantarum strain' LDH sp act Residual L-LDH sp act Total lactate % >-(+)-Lactate(U/mg of protein) (U/mg of protein)' (g/liter)c

NCIB8826[pGIT032] 50.0 ± 6.2" 46.9 ± 4.9d 14.6 ± 0.4" 40NCIB8826[pLAB1301] 6.9 ± 0.5" 3.6 ± 0.3d 16.5 ± 0.4d 47TF101 (AldhL) 3.3 ± 0.2" Not detected 15.6 ± 10.0 93eNCIB8826 (wild type) 8.6 ± 1.1d 5.9 ± 0.8" 15.8 ± 1.2d 50

a The inoculum was grown anaerobically in MRS broth. Plasmid pGIT032 is a multicopy vector derived from pLAB1301 (control vector) bearing the IdhL gene fromL. plantarum; the TF101 strain corresponds to the NCIB8826 strain with a deletion inside the IdhL gene.

Residual L-LDH activity was determined in crude cell extracts after a 3-min heat treatment of the sample (50°C) to inactivate D-LDH.c Lactate concentrations in culture supernatants were determined enzymatically with a commercial E-( - )- and L-(+)-lactate dosage kit (see Materials and Methods).d These data represent average values (from four individual measures) followed by their standard deviations.e The amount of L-(+)-lactate measured in culture supernatant from TF101 corresponds to the concentration of this isomer in fresh MRS medium.

internal region (L2). Campbell-type integration of the vector(involving the Li or L3 region) results in a wild-type L-LDHphenotype. Secondary excision through intrachromosomal re-combination within the other region of homology (L3 or Li)leads to deletion of the internal L2 region of the IdhL gene.The deletion was first created in a pJDC9-borne copy of the

IdhL gene. Removal of the 305-bp HpaI-NruI fragment (L2)inside the open reading frame resulted in a nonfunctional IdhLgene with a 684-bp 5' fragment (Li) and a 446-bp 3' fragment(U). The resulting plasmid (pGIT015) could have been useddirectly as a nonreplicating vector for the first-step integrationin L. plantarum. However, homologous recombination fre-quencies depend on the length of homologous regions, whichare quite short in pGIT015 (684- and 446-bp). The use ofsuicide vectors is also limited by the efficiency of electrotrans-formation, which is also relatively low with L. plantarumNCIB8826 (about 105 transformants per ,ug of DNA). There-fore, an unstable autoreplicative vector was preferred for thefirst-step integration event. The origin of replication fromplasmid pE194 (38) (XbaI-PstI fragment from pGIP711 [17])was inserted in the XbaI-PstI sites of pGIT015. As expected,

pGITO17

Li L2 PL3

FIG. 2. Construction of a deletion in the L. plantarum IdhL gene.Integration plasmid pGIT017 consists of the pJDC9 vector, the originof replication of the pE194 vector (black box), and a truncated copy ofthe L. plantarum ldhL gene (LI and L3 open boxes) deleted from a305-bp HpaI-NruI internal fragment (L2). Campbell-type integrationof pGIT017 into the L. plantarum chromosome via the Li regionresults in a tandem of wild-type and deleted copies of the ldhL gene.Secondary excision by intrachromosomal recombination via the U3region results in a completely stable deletion of the L2 region in thechromosomal ldhL gene. The same chromosomal alteration can alsoresult from first-step integration via the L3 region followed by excisioninvolving the Li region. Asterisks indicate the positions of theoligonucleotides used for screening IdhL-deleted strains.

the resulting pGIT017 vector was able to replicate in L.plantarum but displayed a high segregational instability in thishost.The plasmid pGIT017 was introduced into L. plantarum

NCIB8826 by electrotransformation. One of the transformantsisolated on selective MRS plates was grown in the presence ofantibiotic for about 10 generations. These selective conditionsmaintained the autoreplicative plasmid in the bacterial popu-lation and facilitated the expected integration event. A fractionof this population was further cultivated for about 50 genera-tions in nonselective medium in order to allow segregationalloss of nonintegrated plasmids. At this stage, about 1% of thebacteria still retained the resistance phenotype when platedonto selective medium. For all the clones tested, this pheno-type was stable in the absence of selection, suggesting that theyhad undergone a primary recombination event. One of theseerythromycin-resistant clones was further grown for 30 gener-ations without antibiotic. Intrachromosomal recombinationfollowed by segregational loss of the excised vector led toerythromycin sensitivity which was observed in about 1% of thepopulation. As described above, a secondary excision event canrestore the initial L-LDH phenotype or else result in a deletionin the chromosomal gene. To distinguish between these twopossibilities, a pair of oligonucleotides was chosen on bothsides of the deletion site (Fig. 2, asterisks). In this way, PCRperformed on chromosomal DNA would amplify a 462-bpfragment for the wild-type copy of the gene, whereas thedeleted allele would yield a shorter fragment of 157 bp. A totalof 5 of 12 tested clones yielded the shorter amplified fragment.Sequencing of the 157-bp PCR fragment definitely confirmedthe deletion (data not shown). This L. plantanum strain wasdesignated TFlOl. According to our strategy, the secondrecombination event (excision) could result with equal proba-bility in either wild type or L-LDH deficiency phenotype. Theproportion of deleted clones (5 of 12 candidates) and the factthat the deleted and the control strains have similar growthcurves suggest that the loss of L-LDH activity, and conse-quently the absence of L-(+)-lactate production, is not detri-mental to this bacterium.

Specific LDH activity measurements in crude cell extractsfrom L. plantarum TF101 and NCIB8826 (wild-type strain)after 22 h of anaerobic growth are listed in Table 2. These datademonstrate a 60% reduction in total LDH activity in theIdhL-deleted strain, with no detectable L-LDH activity. Datareported in Table 2 also show that L. plantarum TF101 onlyproduces D-( - )-lactate, and the production of this isomer wasincreased so that the global concentration of lactate in theculture supernatant remained virtually unchanged. This com-pensatory production of D-(-)-lactate by the TF1l1 strainafter 22 h of growth is not due to a late accumulation of this

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600 FERAIN ET AL.

isomer. Indeed, similar results have been obtained at variousstages taken in both the growth phase and the stationary phase(data not shown).

In conclusion, our results show that the amount of pyruvatereduced to lactate in L. plantarum is not limited by the activityof either L- or D-LDH. Indeed, neither the increase nor theabsence of the L-LDH activity in the manipulated strainsresulted in a modification of global lactate production in thisbacterium. These observations, as well as the fact that the ratioof L-(+)- to D-(-)-lactate remained close to unity despite thedrastic overexpression of the ldhL gene, suggest that theconversions of pyruvate to both L-( - )- and D-( - )-lactate arenear-equilibrium reactions. Therefore, two possible explana-tions could be envisaged. On one hand, lactic acid productioncould be limited by the rate of glycolytic flux, the pyruvatebeing reduced to lactate as soon as it is produced. Note that theNAD+ coenzyme is not suspected to be a limiting factor sinceits intracellular concentration in lactic acid bacteria representsa large excess of the amount necessary for the operation of theglycolytic system (31). On the other hand, the rate-limitingfactor could be the lactate excretion step which would there-fore operate at the maximum rate. In this case, the compen-satory production of D-(-)-lactate in the L-LDH-deficientstrain would suggest that the two lactate isomers share acommon, nonstereospecific transport system as previously de-scribed for other species (16, 24).

ACKNOWLEDGMENTS

This research was carried out in the framework of the CommunityResearch Programme 'ECLAIR' with a financial contribution from theCommission (contract AGRE-CT90-0041) and from the DirectorateGeneral for Research and Technology of the Walloon Region (con-tract 1580). T. Ferain and N. Bernard hold an I.R.S.I.A. specializationfellowship.We are indebted to E. Van Schaftingen for stimulating discussions

and comments. We are grateful to J. Mahillon and A. Fitzsimons forcritical reading of the manuscript and to K. Josson for providing thepLAB1301 plasmid.

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