genetically modified lactic acid bacteria: applications to food or health and risk assessment

8/12/2019 Genetically modified lactic acid bacteria: applications to food or health and risk assessment

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cal properties and increase the reliability of food makingprocesses, and improve product safety and quality. More-over, LAB can be engineered to function as cell factories.Cell metabolism can be engineered to massively producemetabolites of interest such as food additives and aromacompounds. They were also shown to be able to produceproteins with applications to health or the development of new vaccines. Lastly, it might be expected that the develop-ment of knowledge of the interaction between certain LABand the human host will allow to better exploit their expectednatural potential to improve health.

2. Potential of genetic technology in LABcloningsystem and engineering strategies

Genetically, LAB may have been the rst organism inwhich genetic exchange was demonstrated since Pneumo-coccus was transformed in 1928 in vivo in mice by Grif th.The transforming principle was found to be DNA in 1944 byO.T.Avery et al. (reported by Avery et al. [5]) and a protocolfor genetic transformation was described in 1957 by Braccoet al. [6]. However, most genetic discoveries in bacteria havebeen made in one or other of the two major model microor-ganisms, Escherichia coli and Bacillus subtili s. A new startin the genetics of LAB, especially those bene cial to man,came from the possibility of producing and regeneratingprotoplasts [7] and nally of transforming them [8]. Newtechniques such as electroporation have been developed,allowing the transformation of most species of LAB [9]. Weshould also mention alternative processes of genetic ex-change in LAB like conjugation and transduction [10]. Most

of these mechanisms initially developed in Lactococci havesince been found to work in many other species of LAB.

The impressive increase in interest in the genetics of LABhas led to the development of a variety of genetic systems toanalyze and modify the metabolism of these bacteria. This isparticularly evident for the species L. lactis that has becomethe paradigm for LAB, but many of these tools are nowbecoming available for other LAB of industrial interest. Tosummarize, these systems may be classi ed as cloning sys-tems, chromosome modi cation systems, and expressionsystems [11]. We will mention here only those that are of socalled food grade and are allowed to be introduced into ourfood. In general, food grade systems have to contain onlygenetic elements that are as safe as the host. It is more or lessaccepted that these elements must have originated from bac-teria that already have a long history of use in food. In mostcases, these genetic elements derive from plasmids and genesfrom bacteria of the same species, to provide a self-cloningsystem , facilitating their agreement foruse as de ned by thenovel foods procedure in the EU. In all cases, they should bewell characterized and not contain antibiotic resistancemarkers, and not require the use of harmful compounds. Inaddition to these safety issues and legal constraints, foodgrade systems have to meet minimum requirements for sta-

bility under industrial conditions and scale of use and have tobe applicable in an ef cient and cost-effective manner.

2.1. Food-grade cloning systems

A number of plasmid vector systems have been developedusing the origin of replication of natural plasmids combinedwith food-grade selection markers, such as the wide host-range pWV01 or pVS40 plasmids [12,13], the narrow host-range pCI305 [14] in Lactococci , or pFR18 in Leuconostocmesenteroides [15]. Integrative plasmid strategies have alsobeen developed using phage or transposon integrative sys-tems, such as those of the A2 phage of L. casei [16], mv4 of L. delbruekii [17] and TP901-1 in L. lactis [18]. Lastly,systems based on homologous recombination by singlecross-over of non-replicative plasmids were used as integra-tive vector by removing part of their replication functions[19,20].

These strategies necessitate the use of selective markers

that will allow their selection and maintenance in the host.Two kinds of markers can be de ned, those that are selectablebecause they confer a new phenotype, and those that restorean impaired function. In the rst class are sugar utilizationmarkers such as sucrose [21] or xylose [22] genes, bacterio-cin resistance genes such as those conferring insensitivity tonisin [23,24] or lactacin F [25] or metal such as cadmium[26]. For the second type of marker, any function necessaryfor cell viability and that can be conditionally inactivated toproduce auxotrophic mutants can be used.A systembased onthe inactivation of lacF , a lactose gene, has been developed in L. lactis [27,28]. A system based on a suppressor tRNAallowing growth in milk of a purine auxotrophic strain was

designed for industrial L. lactis strains [29,30]. Alanineracemase mutants of L. plantarum able to grow only in thepresence of D-alanine were also constructed, and could becomplemented by the functional gene [31,32]. The presentlist is not exhaustive, but shows that a number of food-gradevectors and markers are now available in different LAB.

2.2. Targeted chromosomal modication systems

Although many vectors are now available, the systemsdescribed in the previous section have several disadvantages:(i) copy number of plasmids may vary, (ii) plasmids may belost in the absence of marker selection and (iii) plasmids maybe structurally instable. Moreover, these vectors require theintroduction DNA in addition to that of the desired gene.Tools are also available to insert genetic constructions byallelic replacement in the chromosome. This method hasseveral advantage over replicative or single cross-over inte-gration vectors. In particular, allelic replacement allowsstable DNA insertion or genetic modi cations without leav-ing any foreign DNA other than that desired. Allelic replace-ment occurs by double cross-over between two regions of homology anking the modi cation and the correspondingregions on the chromosome. This proceduremay occur spon-

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taneously upon transformation using the natural competencemachinery, such as those described in Streptococcus pneu-moniae [33] and B. subtilis [34]. However, although thegenome of the sequenced LAB contain the set of genesrequired for competence development ( [4], personal data),procedures to induce these systems have not yet been de-scribed. A thermosensitive plasmid-based system has beendeveloped allowing gene replacement by a two-step proce-dure ( Fig. 1 ). A mutation in the gene encoding plasmidreplication protein of the natural plasmid pWV01 has beenselected, allowing maintenance of the plasmid at 30 C, butnot at 37 C [35]. This plasmid can direct homologousintegration in the L. lactis chromosome when it carries achromosomal DNA fragment of suf cient length (about500 bp ormore; [36]) . After the two step procedure, allowingsequential recombination in the two fragments anking thecentral modi cation (nucleotide change, deletion or DNAinsertion), the thermosensitive plasmid vector with selectivemarkers can be cured by simply growing the modi ed strain

at 37 C for several generations to allow plasmid segregationonce replication is blocked. Lastly, this plasmid can also beused to select food-grade mutants containing a single ISelement as new DNA fragment in the genome [37].

2.3. Expression systems

In addition to cloning systems, gene expression systemshave been developed allowing the controlled expression of homologous or heterologous genes. Most of these systemswere developed in L. lactis, such as those based on promoterscontrolled by sugar (lactose operon promoter, [38]) , by salt(gadC promoter, [39]) , by temperature upshift ( tec phage

promoter, [40,41]) , pH decrease (P170, [42]) and phageinfection (phi31-promoter, [43]) . A dose-dependant systemof induction is also available using sublethal concentrationsof nisin in L. lactis [44] and this system was extended tosome other LAB [45,46]. Sugar-dependant expression sys-tems have also been developed in other LAB such as lacto-bacilli [47,48]. Although very interesting for the productionof heterologous proteins, inducible systems are not alwayseasy to manage under industrial conditions, especially if aconstant level of productionis required for metabolic control,for example. In this case, a well-de ned constitutive pro-moter with the desired level of expression may be moreef cient. A system of synthetic promoters allowing the con-stitutive and de ned level of expression of downstream geneshas been created recently [49] and could, in principle, beapplied to any bacterial species [50].

3. Strain improvement in food technology

Since the rst starter cultures were used for fermenteddairy products, the main stream of research has been per-formed to improve dairy starter strains, and in particular L. lacti s. Nevertheless, in the last few years, there has been a

considerable increase of interest in the other species of LAB,including those involved in meat, vegetable and wine fer-mentation. In this section we will describe theconstructionof improved starter strains from the most simple designs tomore complex variants.

3.1. Producing genetic variants

LAB are naturally diverse and strains belonging to thesame species may have very different properties. These dif-ferences are largely exploited, offering a wide range of strains that can then be combined as a function of the re-quired processes and products. However, one may want tocombine a speci c trait of one strain with another strain thathas a background well adapted to a particular process. Thiscould be of particular importance if only a single strain isavailable for a given process, precluding the use of alterna-tive strains in case of phage attack, for example (see belowfor phage issue). There is thus a need to be able to combine

strain characters in order to produce reliably fermented foodproducts of high quality.

In some cases, the desired modi cation could be restrictedto the mutation of a single gene, a process that can occurspontaneously, sometimes at relatively high frequencies.Thus, lactococcal variants in lactose metabolism [51], citrateuptake [52] and proteolytic activity [53] can easily be ob-tained by simple screening procedures, because the genesnecessary for these metabolic pathways are encoded on seg-regationally unstable plasmids. These variants can be se-lected at frequencies ranging from 10

3 to 101 .

However, the number of traits that could be modi ed bysuch an easy method is quite limited, and more ef cientscreening strategies should be set up to select mutants arisingat frequencies of 10

6 or lower, which is approximately thelevel of spontaneous mutation of a gene in the chromosome.In some cases, screeningcanbe facilitated by color reactions,such as the use of X-gal to select strains devoid of b-galactosidase activity. This screening strategy was used toselect L. bulgaricus strains unable to ef ciently fermentlactose and do not acidify yogurt after fermentation hasnished [54]. Indeed, upon storage, yogurt pH may dropbelow a value of 4.0, increasing the acid and bitter taste of theproduct, and thus degrading its initial organoleptic quality.This post acidi cation process is mainly due to L. bulgaricuslactose metabolism. Lactose de cient strains are still able togrow in association with S. thermophilu s, the second yogurtstarter strain. Use of such a strain allows the production of yogurt that can be kept for months at 4 C without a signi -cant drop of the pH [55].

Searching for natural variants is, in principle, possible forany gene as long as its function is not essential. However, itcould be dif cult to isolate the proper mutant among millionsof wild type cells, especially for spontaneous mutations thatare not stable as it is the case when it confers a slight decreasein growth rate. Use of mutagenic compounds such as EMS or N -methyl- N '-nitro- N -nitrosoguanidine may be used to in-

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crease the rate of recovery of mutations. LDH mutants hav-ing a mixed pattern of fermentation, and producing increasedamounts of acetoin and diacetyl, were selected by such mu-tagenesis strategy [56]. However, additional mutations may

occur necessitating careful testing for other important traits.To circumvent this problem, genetic engineering can be usedadvantageously. Improving the avor and the avororal sta-bility in buttermilk through metabolic engineering of L. lac-

Fig. 1 . Food-grade allelic exchange and gene integration in the chromosome by a two-step procedure using a thermosensitive plasmid.

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tis subsp. diacetylactis may be presented as a paradigm forthis issue in LAB.

Diacetyl is responsible for the butter avor in many freshdairy products such as butter, cream and buttermilk. How-ever, even if its presence at low concentration is suf cient toconfer this typical aroma, diacetyl is highly labile and its lossresults in a at taste of the products. The main pathway fordiacetyl production is a two step synthesis from pyruvate.The rst step is common to valine biosynthesis and acetoincatabolism pathways through the reaction of two pyruvatemolecules to give a -acetolactate catalyzed by a -acetolactatesynthetase. Diacetyl is then formed by a chemical oxidationoccurring spontaneously at low rate. However, in L. lacti s,a -acetolactate is also actively decarboxylated by a -aceto-lactate decarboxylase into acetoin, a compound that does notconfer the desired avor ( Fig. 2 ). Inactivating aldB, the gene

encoding a -acetolactate decarboxylase, should thus increasethe availability of a - acetolactate for chemical oxidation. Anexperimental protocol to isolate spontaneous aldB mutantswas designed with prototrophic L. lactis strains taking ad-vantage of the misbalance in the ux of acetolactate betweenvaline synthesis and acetoin catabolism in the presence of leucine [59]. As this screen can only be applied to valineprototrophic strains and that most dairy starter strains are not[63], the inactive valine biosynthesis genes were comple-mented with pMC004, a plasmid carrying the ilv operon(Fig. 3 ; [60]) . After selection of the desired aldB mutation,pMC004 was removed leading to a mutant that did notcontain any foreign DNA. This strain accumulates highamounts of a-acetolactate leading to stable formation of diacetyl throughout 3 weeks of storage. Currently, this strainis used in combination with a diacetyl reductase Leuconostocmutant in a starter commercialized in the USA.Although theDanish Veterinary and Food Administration granted permis-sion to use this strain during an international Dairy meeting

in the EU, this strain is still considered as a GMO and is notused in the EU [62]. Issues concerning the use of GMOs inthe EU will be discussed at the end of this paper, in thesection on risk assessment.

Gene technology allows the production of similar aldBmutants by direct allelic replacement using an appropriatethermosensitive vector [61]. This approach was also adoptedto obtain food-grade mutants of L. lactis resistant to phagesby the inactivation of the phage infection protein ( pip)involved in phage adsorption and DNA injection [64]. Simi-Fig. 2 . Pyruvate metabolism leading to lactate, alanine, leucine, valine and

aromatic compounds.

Fig. 3 . Different procedures to produce mutants inactivated in the aldB gene encoding acetolactate decarboxylase. Left column: classical mutagenesis and alaborious screening to isolate an aldB mutant [57], central column: the aldB mutant is selected by a screen using the dual role of L. lactis acetolactatedecarboxylase in the regulation of the acetolactate pool ( [58], Fig. 2) to select leucine resistant clones (Leu R ) that do not display growth inhibition in achemically de ned medium containing leucine but not valine and isoleucine [59]. A plasmid carrying the ilv operon should be provided to valine auxotrophicstrains [60]. Right column, two-stepprocedure to exchange the functional aldB allelefor an inactivatedcopy of thegene [61]. The rst procedure leadsto strainsthat do not need to be labeled as GMO, while the two procedure involving plasmid transformation lead to GMOs [62].



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lar approaches could also be used in other LAB species suchas S. thermophilus where the inactivation of the phosphoglu-comutase gene enhances polysaccharide production [65] andthat of urease genes reduces delay in the acidi cation inmilks containing high amount of urea (Anba et Renault, inpreparation).

Lastly, speci c mutant may be isolated by use of an ISelement [37]. The nal strains only contain ISS1 originatingfrom L. lactis at a well de ned place in the chromosome, forexample in the target genes presented above. Interestingly,ISS1 mutants affected in complex genetic and physiologicalresponse were obtained, such as stress resistant strains af-fected in guaA (inactivation), relA (modi ed activity, [66] ) ormutant in proteolysis [67,68]. In both cases, it is expectedthat the expression of a number of genes is affected. In therst case, it is proposed that the increased resistance to stressis due to a decrease in the intracellular GTP pool and/orstringent response, a response involving a drastic change inthe cellular metabolism and regulation in E. coli [69] and

B. subtilis [70]. However, relatively little is known aboutthese intracellular controls in LAB or about the real rel-evance of such mutants in fermentation. In the second case,the mutants are inactivated for CodY, a regulator initiallyfound to regulate early-stationary-phase genes in B. subtilis[71]. In L. lacti s, this regulator was found to regulate themost important genes involved in casein utilization. In codY mutants, the transcription of the cell-wall protease genes, apeptide permease operon and the major peptidase genes isincreased between 5 and 100-fold. In addition to these genes,it is now known that CodY also regulates at least someaminotransferases (Yvon, personal communication). Sinceproteolysis and amino acid degradation have a major role in

cheese ripening [72], it is expected that codY mutants willhave some interesting properties for cheese making and thepotential interest of codY mutants is now being tested.

3.2. Constructing modi ed strains with genes from other LAB

In the precedent cases, the modi ed bacteria could beconsidered as mutants equivalent to bacteria that might al-readyexist naturally. However, introducing newDNA encod-ingnew information into thebacterialcell could lead to widerstrain diversity. In the present section, we will examine ex-amples of engineered strains obtained by the introduction of new genes from other bacteria of the same or a differentspecies of LAB. In the rst case, the modi ed strains are byself-cloning, while in the second, the notion of self-cloningrequires discussion (see the section concerning risk assess-ment).

To obtain strains with increased proteolytic properties, thegenes encoding PepN, PepC, PepX and PepI peptidases of ahighly proteolytic L. helveticus strain [73] or PepI, PepL,PepW, and PepG from L. delbrueckii [74] were transferredinto L. lactis using a food-grade cloning system. It is ex-pected that such recombinant bacteria producing an addi-tional peptidolytic enzyme activity may make an important

contribution to proteolysis during maturation of cheese, forexample, by shortening the ripening period and allowing theproduction of special cheeses (e.g. reduced-fat cheeses) withimproved characteristics.

Another example of gene transfer between LAB is pro-vided by the construction of bacteriophage resistant strains.Indeed, LAB that are used repetitively and massively inindustrial productions can be highly subject to infectionswith bacteriophages. This leads to the lysis of the starter andthereby the arrest of fermentation. The products obtainedthen do not have the desired quality, and may eventually belost. The origin of bacteriophages is still discussed, sincethey may come from raw products such as milk that hadcontact with environmental farm bacteria including wildLAB, from inoculum (mostly if it is not properly produced),from the factory itself, or from the evolution of remnantphages present in the starters. Selection of bacteriophageresistant strains is thus an ongoing task of starter producers.Research on bacteriophage resistance determinants in certain

strains led to the characterization of a number of resistancemechanisms (recently reviewed by Forde and Fitzgerald,[75]) . Phage resistance systems may interfere with phageadsorption, phage DNA injection, phage replication, tran-scription, RNA translation, protein assembly and phagepackaging. These mechanisms are often carried out by mo-bile elements such as plasmids and transposons suggestingthat lateral transfer of these genes occurs under pressure of phage infection. Some high level resistance plasmids wereshown to carry more than one resistance mechanisms. Toimprove phage resistance, one could rationally combinethese mechanisms as a function of their target in phagedevelopment and of the phages present in the factories. A list

of mechanisms, with emphasis on those patented has beenpresented in a recent review [76].

In addition to these natural resistance mechanisms, recentadvances in the knowledge of phage biology has allowed thegeneration of new weapons by targeting speci c steps inphage development. For example, to lower phage prolifera-tion, it has been proposed to introduce a further phage repli-cation origin that competes with that of the phage [77].Another strategy is to induce the expression of a lethal geneupon phage infection [78] or to massively produce antisensemRNA against essential phage genes [79,80]. The most im-portant drawback of these systems is their narrow range of action. Lastly, DNA shuf ing, exploiting the properties of atype I restriction enzymes could also generate newrestriction/modi cation mechanisms [81].

Phage and cellular genes involved in cell lysis were pro-posed to be used to construct cells that will lyse at an appro-priate moment during cheese making to improve cheeseripening. Indeed, whereas starter lysis is a major problemduring fermentation, late cell lysis allows the release of manyenzymes into the cheese matrix, improving in particular thedegradation of peptides.This degradationallows cheese to bemade less bitter (due to the reduction of some bitter peptide),and contain more free amino acids (precursors of aroma).



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Several approaches have been proposed to provoke con-trolled lysis of starter bacteria, including the use of autolyticstrains, bacteriocin producing starters [82] and phages [83].Several of these approaches are not easy to optimize, or mayeven be seen as unacceptable by industry because of the useof phages that may contaminate other processes in the fac-tory. Engineered strains producing phage derived lysin andholin proteins [84] or bacteriocin [85] under the control of aninducible promoter have been constructed. Increased lysis of the cells was obtained and cheese trials have shown thatunder some conditions this lysis may allow an increasedaroma production. However, additional work is needed tooptimize the strains for industrial use. In addition to acceler-atingcell lysis for cheese ripening, heterologous bacteriocinscould also be produced to eliminate undesirable contaminantbacteria [86].

Lastly, we will brie y mention two examples where newfunctions are provided by inter LAB cloning. An amylolytic L. plantarum silage strain with high starch-degrading ability

was developed by expressing the L. amylovorus amylasegene. This recombinant strain may have potential as a silageinoculant for crops such as alfalfa, in which water-solublecarbohydrate levels are low but which contain starch as analternative carbohydrate source [87]. As a second example,an L. lactis strain containing the complete eps cluster fromS. thermophilus S6 is able to produce an exopolysaccharideof similar size to that of the native strain. However, itscomposition differs suggesting that an additional chromo-somal copy was required for its complete synthesis [88].Similar experiments carried out with the eps cluster fromS. thermophilus S39 allowed the production of an EPSsimilar to the one produced by S 39 [89]. These examplesshow that complete complex biosynthesis pathways can beintroduced in LAB, and in the above mentioned case, wouldhave application to feed production or in the improvement of the texture of fermented food.

3.3. Modi ed strains with heterologous genes

A number of applications can be proposed that depend onmodifying genes or transferring genes from food LAB intoother lactic starter strains. However, some functions may notexist in LAB, and such modi cation would necessitate thetransfer of DNA from more distant bacteria.

As a model, a heterologous catabolic glutamate dehydro-genase (GDH) gene from Peptostreptococcus asaccharolyti-cus was introduced into L. lactis to allow this organism toproduce alpha-ketoglutarate from glutamate, an amino acidpresent at high levels in cheese. Indeed, during cheese ripen-ing amino acid degradation plays a major role and its rststep in lactococci is a transamination, which requires analpha-keto acid as the amino group acceptor [90]. Moreover,the rst limiting factor for conversion of amino acids toaroma compounds may be the level of available alpha-ketoacids. Interestingly, the GDH-producing lactococcal strainproduced a higher proportion of carboxylic acids, which aremajor aroma compounds suggesting that such modi ed

strains could be used to avoid alpha-ketoglutarate supple-mentation [91].

Another interesting example of genetic engineering is theredirection of metabolism to produce high added value prod-ucts. For example, it was proposed that LAB such as L. lactiscould be used to produce high amounts of L-alanine notcontaminated by the D-stereoisomer [92]. Alanine could beproduced from pyruvate in a single step by alanine dehydro-genase ( Fig. 1) . To redirect the carbon ux from pyruvate(which usually leads to lactate) to alanine, the Bacillussphaericus alanine dehydrogenase was expressed in anL-LDH-de cient lactococcal strain. The constructed strainproduced alanine as the sole end product. Finally, stereospe-cic production (>99%) of L-alanine could be achieved byinactivating the host-gene encoding alanine racemase.

Although of great potential interest, the use of LAB modi-ed with exogeneous genes may be limited, at least in thenear future, by the suspicion consumers may have concern-ing the use of such genetically modi ed organisms alive in

food. We will present in the next section, several other ex-amples of gene transfer technology in LAB directed to thera-peutical applications for which public perception is morepositive.

4. Potential application of LAB to improve health

The consumption of speci c LAB, mainly members of Lactobacillus, has been proposed to be bene cial for humanhealth, by the prevention of gastrointestinal tract infections,by immune stimulation, and by the balancing of intestinalmicro ora. These potential health promoting bacteria areoften called probiotic . It is thus tempting to propose re-search work in order to select and modify these microorgan-isms to improve their properties or confer on them new ones.However, the molecular mechanisms underlying probioticcharacteristics often remain controversial and furtherprogress concerning the molecular basis of probiotic traitswill be a prerequisite for the rational development of furtherapplications [93,94]. Interestingly, other approaches to theuse of LAB for promoting health wasproposed by modifyingstrains that initially had no known probiotic effect. In thiscase, the term probiotic should probably be avoided sincethese bacteria were constructed for direct medical applica-tion and not for distribution to consumers (in food or by othermeans) without medical control. We will focus our attentionon these applications in this section.

Progress in medical science allows the design of strategiestargeted to cure disease, including drug delivery, the supplyof molecules compensating defects or the stimulation of human functions such as the immune system. Some of thesestrategies make use of molecules that could be synthesizedby bacteria, including LAB. Most work in this domain hasbeen devoted to the development of new vaccine strategies,but has also concerned substitution therapy to correct en-zyme defects in the digestive tract [95].



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4.1. Vaccine development and modulating the immunesystem

Mucosal routes for vaccine delivery offer several advan-tages over systemic inoculation by minimizing potential ad-verse effects and by the ease of administration. One way todeliver protective antigens at mucosal surfaces is to use livebacterial vectors. Over the last two decades, the use of re-combinant bacteria as carrier system to deliver antigens tothe mucosal immune system has been widely investigated.Most strategies have relied on the use of attenuated patho-genic bacteria, among which is the use of invasive but non-pathogenic Salmonella [96]. A number of other bacteria suchas Listeria, Vibrio, Bordetella, Mycobacterium have alsobeen proposed, although the major concern in the use of attenuated pathogens is that they may still be virulent in theelderly and in very young children. Food LAB usage wouldovercome this problem since these bacteria have a longhistory of safe use and could possibly be delivered safely at a

high dose.Many antigens have been expressed in LAB such as L. lactis and L. plantarum , but also in the human commensalStreptococcus gordonii , mainly within the framework of several European programs (reviewed by Mercenier et al.[97]) . A selection of various studies is presented in Table 1 .Among the rst and most extensively studied antigens isfragment C of tetanus toxin, which has been massively pro-duced in L. lactis and shown to protect mice immunizedsubcutaneously against lethal challenge [98]. Further work showed that it was possible to protect mice against tetanus

toxin by nasal [99] and oral [100] fragment C administrationand that other LAB could also be used for this purpose [101].Vaginal immunization could be induced by the use of S. gor-donii [105 107]. In addition to protein antigens, some anti-genic polysaccharides could also be synthesized by LAB[109]. Moreover, the immune response can also be potenti-ated by co-expression of interleukins such as IL-2 and IL-6[114]. Other cellular mediators such as IL-10 could contrib-ute to the treatment of in ammatory bowel diseases [112].Studies have been initiated to better understand allergy andeventually modulate responses to food allergens [115].

Lastly, new strategies have been proposed against experi-mental candidiasis by the expression of an anti-idiotype inS. gordoni i. This human commensal bacterium was engi-neered to locally release a microbiocidal single-chain anti-body. The bacteria stably colonized rat vagina and allowedthe treatment of experimental vaginitis caused by Candidaalbicans [118,124]. The same team also expressed anotheranti-idiotypic single chain fragment variable (scFv) recom-binant antibody to vaccinate against group B streptococci[119]. A similar approach led to the construction of Lactoba-cillus zeae producing ScFv at their surface in order to ghtagainst S. mutans involved in dental caries [125].

Although these approaches are promising, many ques-tions remainunanswered.Among these are theecology of thecarrier LAB that could persist in the host or be rapidly lost.Biologically contained strains could be the goal of futureimprovements [97]. Methods to tag bacteria or to study theirphysiology upon administration are now available for this

Table 1Examples of studies showing the potential of LAB to be engineered for therapeutic application

Organism Molecule Main objectives Reference L. lactis L. plantarum Fragment C of tetanus toxin Protection against tetanus toxin [98-101] L. lactis L. plantarum Fragment C of tetanus toxin Study the effect of epitope location for vaccin delivery

by LAB[102,103]

L. plantarum Model antigen M6-gp41E (human immunode ciencyvirus gp41 protein)

Vaginal immunization for HIV [104]

S. godonii, L. casei V3 domain of the gpl20 of human immunode ciencyvirus type 1 (HIV-1)

Vaginal immunization for HIV [105 107]

S. godonii, L. casei E7 protein of human papillomavirus type 16 (HPV 16) Vaginal immunization for papillovirus [105,106] L. plantarum Cholera toxin B Protection against cholera toxin B [108] L. lactis Pneumococcal type 3 capsular polysaccharide Mucosal immunization against Streptococcus

pneumoniae[109]

L. lactis Bovine rotavirus nonstructural protein 4 Protection against rotavirus diarrhea [110] L. lactis Brucella abortus ribosomal protein L7/L12 Vaccine against brucellosis. [111] L. lactis Murine interleukin-10 Treatment of in ammatory bowel diseases [112,113] L. lactis Murine interleukin-2 and 6 Enhancement of immune responses [114] L. lactis Bovine beta-lactoglobulin Modulation of immune responses to food allergens [115] L. lactis Staphylococcus hyicus lipase Compensation of pancreatic insuf ciency [116,117]S. godonii Microbicidal single-chain antibody (H6) C. albicans vaginitis [118]S. godonii Anti-idiotypic single chain fragment variable

recombinant antibody mimicking the type III capsularpolysaccharide of group B streptococci

Passive protection of neonatal pups from group Bstreptococci disease

[119]

S. godonii M6 protein of S. pyogenes Antigen delivery system [120] L. lactis S. aureus protein A Antigen delivery system [121] L. lactis S. aureus nuclease Antigen delivery system [122] L. lactis L. bulgaricus proteinase Antigen delivery system [123]



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purpose [126,127]. Moreover, it has been suggested thatantigen presentation may be of crucial importance in immu-nization, and that certain constructs could induce immuno-tolerance rather than immunization [128]. Different tools arenow available to present antigens in a different way and suchissues could be tested in model systems [98,99,103,113,122,129]. Possible limitations due to secretion machin-ery bottlenecks are also under investigation [130 133].

4.2. Compensation for metabolic defects

The potential of LAB to correct metabolic defects hasbeen relatively less studied. However, it hasbeen known foralong time that fermentation by LAB reduces lactose intoler-ance [134]. Although it is controversial, it hasbeen suggestedthat bacterial lactase could naturally supplement the humanenzyme in cases of de ciency. The idea that LAB couldcompensate for enzyme de ciency is thus not new. Recently,lipase from S. hyicus was produced massively intracellularlyin L. lactis in order to deliver high quantities of this enzyme

in the jejunum [116]. Indeed, L. lactis was shown to be able,under certain conditions, to pass through the stomach andmassively lyse in the jejunum where the lipase should bedelivered [126]. Oral treatment with L. lactis expressing thislipase was carried out in a pig model where pancreatic insuf-ciency was induced by ligation of the pancreatic duct. Thecoef cient of fat absorption was signi cantly higher afterconsumption of lipase-expressing L. lactis than after con-sumption of the control strain showing that this strategycould in principle be applied to compensate for enzymedeciency [133].

5. Other applications

In addition to food and health applications, geneticallymodi ed LAB have been used as improved biosensors for thedetection of biocides in milk. Initially, a commercial productmade use of a S. thermophilus strain particularly sensitive toantibiotics to detect possible contamination of milk thatmight perturb fermentation. This test requires a few hours toassess whether the metabolic activity of the strain is inhib-ited. To shorten it, luciferase genes were introduced in thisstrain and their expression optimized [135]. Finally, usingreduced light production in highly bioluminescent S. thermophilu s, test times could be signi cantly shortened comparedto the previous commercial test utilizing the related non-bioluminescent strain. This GMO, presenting an improvedtest kit to detect antibiotic residues in milk (Valio Oy), is theonly LAB which was approved under the directive90/220/EEC since December 1997. It should be mentionedthat this bacterium is not present in food products and isdestroyed after the test has been realized on a small sample.

6. Risk assessment

Fermentation-based bioprocesses rely extensively onstrain improvement for commercialization. Until now, strain

improvement was mainly based on the selection of newnatural strains but, as detailed in the previous sections, nu-merous improvements can be obtained by using gene tech-nology. However, at least in Europe, no modi ed LAB arecommercialized as yet, and some aspects underlying thisissue will be discussed here. Community legislation onGMOs has been in place since the early 1990s, but thisregulatory framework has been further extended and re ned.It was designed to protect the health of EU citizens and theenvironment at the same time as the creation of a uni edmarket.

6.1. EU legislation and risk assessment procedure

To date, Directive 90/220/EEC is the main legislationauthorizing experimental and commercial release of GMOsin the Community. An updated Directive 2001/18/EC on thedeliberate release of GMOs should apply on 17 October2002. Directive 90/220/EEC put in place a step-by-step ap-proval process on a case-by-case assessment of the risks tohuman health and the environment before any GMO or prod-uct consisting of or containing GMOs can be released intothe environment or placed on the market. Products derivedfrom GMOs are covered by the Regulation on Novel Foodsand Novel Food Ingredients (258/97). Lastly, Directive90/219/EEC amended by directive 98/81/EC regulates thecontained use of GMMs for research and industrial purposes.

The objective of risk assessment is to identify and evaluatepotential adverse effects of GMOs. These effects could beeither direct or indirect, immediate or future. The cumulativeand long term effects on human health and the environmenthave also to be taken into account. The risk assessment looksspeci cally at how theGM product wasdeveloped andexam-ines the risks associated with thegene products in theproduct(for example toxic or allergenic proteins), but also after apossible gene-transfer (for example antibiotic resistancegenes). The determination of the overall risk of the GMOsrequires the identi cation of any characteristics of the GMOswhich might cause adverse effects, the evaluation of theirpotential consequences and their possible occurrence. Fromthese data, the risk can be estimated and the application of management strategies for risks from their use may be re-quired.

6.2. Is regulation the real problem?

Most discussions about the use of GMOs in EU relate tothe use of GM plants and the most recurrent issue raised byopponents is our lack of knowledge of the effect of potentialgene transfer to the environment. However, test experimentsset up by governmental institutes to measure gene ow arealso often the target for highly mediatized illegal destruction.Many opponents of GMOs demand a better distribution of the resources of the planet, on the underlying principal thatGMOs are an industrial advance that pro t only a smallfraction of the population. Although most of these issues arenot relevant for microbial GMOs, the climate of mistrust



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covers all GMOs, except perhaps those targeted to medicaluses. These social issues are far beyond the scope of thisreview, and to summarize, it is possible to obtainapproval forthe use of GMOs in the EU, but the subsequent use of theseGMOs in the open market is hampered by the mistrust of consumers and a strict labeling legislation that allows theconsumer to select products as a function of their content inGMOs (90/220/EEC and Council regulation 1139/98) orderived products (Regulation (EC) 50/2000).

6.3. Strain improvement and safety: GM LAB against natural strains?

Although it will be a determinant factor for the future useof GMOs in the EU, we will not speculate about the evolutionof consumer attitudes in this section. We will rather discusswhat could/should be or not be considered as a GMO andlabeled as such. Indeed, in the EU GMOs (animals, plantsand microorganisms) are de ned as organisms in which thegenetic material has been altered in a way that does not occur

naturally by mutation, mating or natural recombination. Ge-netic engineering technology allows selected individualgenes to be transferred from one organism into another, evenbetween non-related species. Is this de nition relevant in thelight of current scienti c knowledge including, in particular,genomics? If we consider that safety issues should predomi-nate in the GMO debate, what would be the safest procedurefor strain improvement?

First, it should be underlined that the GMO de nition isnot applied similarly in all EU countries: indeed, is the nalconstruction only relevant or should the way in which themodi cation was done also be considered? If we take as anexample the diacetyl overproducing strains, the same muta-tion inactivating the aldB gene could in principle be obtainedin different ways, i.e. spontaneous mutation, induced mu-tagenesis, or genetic engineering (see 3.1 and Fig. 3) . InDenmark a spontaneous mutant isolated after a transient stepin which the strain contained foreign DNA has been consid-ered as a GMO, although this strain should be similar to andpossibly even less modi ed than one obtained by chemicalmutagenesis [62]. Also, a mutant obtained by allelic replace-ment of aldB by a modi ed copy of the gene, even if themutation is a deletion similar to a natural event would beconsidered as a GMO. This is due to the fact that in additionto the nal product (that the US regulation takes only intoaccount), the EU regulation considers to be relevant theway the modi cation was performed. In terms of safetyevaluation, the means by which a mutant has been con-structed should not be relevant unless the technique itself introduces side effect. Of the latter, very little is known yet,except that induced randommutagenesis by UV or chemicalsoften produces secondary mutations due to the lack of target-ing of the method. On the other hand, genetically drivenmutagenesis is considered to allow targeted modi cationsand should thus avoid additional mutations. However, theformal demonstration of the directness of the latter techniquehas not been implemented, especially because until recently,

side effects could be determined only with dif culty due tothe lack of appropriate technology. An EU founded program,Express-Fingerprints (QLK3-2001-01473) is presentlytesting the potential side effects due to the technology in L. lacti s. For this purpose, aldB and pip mutants obtained bychemical mutagenesis and gene technology willbe comparedby twoglobal analytical methods: 2D gelelectrophoresis andtranscription pro ling with DNA microarrays. These meth-ods will allow the determination of relevant changes (de-crease or increase) in the level of expression of at least 450cytoplasmic proteins and of the transcription of over 2100 L. lactis IL1403 genes larger than 89 bp. In addition topinpoint possible side effects, Express-Fingerprint work should also point out the deregulation due to the wantedchange itself. Global expression pro les will thus allow (i) todetermine if changes in addition to those expected occurred,and (ii) to increase our knowledge of gene function.

In addition to variants or mutants of technological rel-evance for or quality improvement, strains expressing genes

originating from genetically closely related species or frommore distantly related species could be developed. In thatcase, at least three issues should be addressed: (i) is theproduct of the new gene(s) safe, (ii) does the new gene(s)induce undesirable functions in the new host and (iii) is thereany danger in case of transfer of the new gene? The rst andthe last issues should be examined case by case, while thesecond could be assessed by the same strategy as that pro-posed by Express-Fingerprints. Several strains of L. lactisexpressing heterologous proteins will be tested in order toevaluate the risk linked to the second hypothesis. Neverthe-less, the main issue for bacteria containing foreign genes,especially those of human origin, is the evaluation of the

potential risk for human health of uncontrolled product ex-pression following transfer of the transgene into a commen-sal bacteria, for example.

An important issue should also be considered if the con-struction technology itself is not considered to in uencesafety. Indeed, whereas it could be reasonably consideredthat many mutants (punctual, insertion of IS, deletion) aresubstantially equivalent to strains naturally occurring (forexample, if a speci c mutation is expected to occur at a verylow rate, i.e. 10

9 per generation, more than 100 such cellswill be present in a yogurt containing 10 9 viable cells per g),it could be asked what additional genes could be addedwithout giving the GMO label? As previously shown, manyof the genes transferred to improve the technology or qualityof a given starter strain are shed from a pool of genespresent in the same or less closely related species. We havenow evidence of many similar transfers in nature; further-more, gene shuf ing occurs on the chromosome[81,136,137], on plasmids [138,139], on transposons [140]or on phage related elements [141,142]. Establishing thelimit of the natural gene pool might not be a trivial issue.

Unfortunately, transfers including undesirable genes mayoccur naturally between bacteria from different species[143]. New genome plasticity data show that strains in the



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same species may signi cantly differ [144,145]. Most of these studies were done on pathogenic bacteria, but majorevolutionary mechanisms might be similar in pathogenic,environmental and food bacteria. Gene transfer has beenshown between pathogenic and commensal bacteria [146].Vehicles for these transfers include phages that have beenshown to be related between pathogens and food bacteria[147]. Phagesarealso known to be vehicles forpathogenicityislands in the former bacteria [148,149]. Therefore, naturaltransfer of genes between pathogenic and natural isolates of food species cannot be excluded. Importantly, food bacteriaare often derivatives of environmental and commensal bacte-ria [150], in particular probiotic strains that are isolated fromintestinal ora [151,152]. Thus, how reliable is the assump-tion that a function isolated from a strain belonging to abacterial species that has a long history of safe use will besafe in another background?

Assessment by global analyses such as done in theExpress-Fingerprints program on a set of natural strains will

only underline the variation in gene expression of genes andfunctions already characterized during the annotation of thegenome and used to design the DNA-microarrays. Any newDNA fragment, and its expression products will not be de-tected (except possibly by proteome analysis) although itcould encode new traits. It might thus appear that GM micro-organisms aremuch bettercharacterized than new strains of agenuine food species. However, one could also expect thatnew high-throughput genomic technologies will allow therapid characterization of new DNA elements present in newstrains. These technology could eventually also be used tocontrol the use of GMO strains, allowing a better character-ization of new strains not included yet in the novel food

regulation.

7. Perspective: how relevant is the concept of GMO?

New technologies such as genome shuf ing are nowemerging [153]. Strain produced by these methods may notbe considered as a GMOalthough thederived bacteria will beless well characterized than tailored GMOs. Genome shuf-ing was successfully applied to improve the acid toleranceof a poorly characterized industrial strain of Lactobacillus[154]. In the rst step, classical strain-improvement methodswere used to generate populations with subtle improvementsin pH tolerance. In the second step, these modi cations wereshuf ed by recursive pool-wise protoplast fusion. Newshuf ed lactobacilli that grow at lower pH or produced morelactic acid than does the wild-type strain were identi ed. Theauthors conclude that genome shuf ing seems broadly usefulfor the rapid evolution of tolerance and other complex phe-notypes in industrial microorganisms. It should be added thatgenome shuf ing is a naturally occurring process using thenatural cellular machinery, such as competence, as shown inpathogenic Streptococcus species. Genome analysis on foodbacteria shows that the full set of genes necessary for thisprocess is present in the completed genomes of L. lactis [4]

and S. thermophilu s, a food bacteria closely related to thehuman commensal S. salivarius (Bolotine and Renault, per-sonal data). The concept that GMO are de ned as organismsin which the genetic material has been altered in a way thatdoes not occur naturally by mating or natural recombinationmay be seen as obsolete in the near future. Moreover, studyof genome evolution will probably provide us with numerousexamples of organisms in which the genetic material hasbeen altered in ways far more complex than man will be ableto mimic.

Acknowledgements

Part of the work for this review was done within theframework of the Express-Fingerprints program (QLK3-2001-01473) of the European Commission. We thank JanKok and Richard H. Buckingham for their careful reading of the paper.

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genetically modified lactic acid bacteria: applications to food or health and risk assessment

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