Genetically Modified Lactic Acid Bacteria: Applications To Food or Health and Risk Assessment

Genetically modied lactic acid bacteria:
applications to food or health and risk assessment

Pierre Renault *
Gntique microbienne, Inra, domaine de Vilvert, 78352 Jouy-en-Josas, France
Received 2 September 2002; accepted 21 November 2002
Abstract
Lactic acid bacteria have a long history of use in fermented food products. Progress in gene technology allows their modication by
introducing newgenes or by modifying their metabolic functions. These modications may lead to improvements in food technology (bacteria
better tted to technological processes, leading to improved organoleptic properties{), or to new applications including bacteria producing
therapeutic molecules that could be delivered by mouth. Examples in these two elds will be discussed, at the same time evaluating their
potential benet to society and the possible risks associated with their use. Risk assessment and expected benets will determine the future use
of modied bacteria in the domains of food technology and health.
2002 ditions scientiques et mdicales Elsevier SAS and Socit franaise de biochimie et biologie molculaire. All rights reserved.
Keywords: Lactic acid bacteria; Food-grade; Biotechnology; Strain development; Risk assessment; Biosafety; GMO; GMM.2
1. Introduction
Lactic acid bacteria (LAB) have a long history of use by
man, and may have been used for food production and pres-
ervation for as long as yeast [1]. These bacteria are gram-
positive, non-spore forming and naturally present in media
rich in organic products such as food products, the digestive
tract, vegetal{. They require a number of growth factors the
nature of which varies among species. They have no respira-
tory system and rely mostly on fermentative metabolism to
provide energy. LAB are, however, a genetically diverse
group of bacteria with GC contents varying from 34 to 53%,
encompassing rod shaped bacteria such as lactobacilli and
cocci such as streptococci, lactococci, entococci, pediococci,
leuconostoc [2]. Moreover, although many representatives of
LAB are perfectly safe and used for generations in food,
some species are pathogens such as pathogenic Streptococci
[3].
Due to the considerable economical importance of LAB,
many groups are now actively working on these bacteria
using an array of genetic tools. Many chromosomal genes of
interest have been characterized. Lactococcus lactis IL1403
was the rst LAB to have been completely sequenced [4],
providing a newinsight into the genetic organization of LAB.
Recently, the genomic sequence of S. thermophilus was
completed in our laboratory in cooperation with Integrated
Genomics and the Universite Catholique de Louvain, and
that of Lactobacillus delbruekii susp. bulgaricus in coopera-
tion with Genoscope. Several other genomes are in the course
of completion at INRA, such as those of L. sakei, L. casei
and Oenococcus oeni. Other sequenced genomes include
L. plantarum (WCFS), L. johnsonii (Nestl), L. acidophilus
(Environmental Biotechnology Institute), L. gaserii, O. oeni
and L. mesenteroides (Joint Genome Institute). This infor-
mation will lead to a better understanding of the physiology
of LAB, in particular by the application of new genomic
technologies such as proteomics, global transcription analy-
sis and comparative genomics. It may be expected that in-
depth understanding of the genetics and physiology of these
bacteria will give rise to new working hypotheses and facili-
tate strain use, selection and improvement.
In this review, we will consider a variety of applications,
already realized or expected to be implemented in the near
future, resulting from the application of life technologies to
LAB. As mentioned earlier, LAB are widely used in starter
cultures in fermented food processing, and a number of
applications have been proposed to enhance their technologi-
* Corresponding author. Tel.: +33-1-3465-2527; fax: +33-1-3465-2521.
E-mail address: renault@jouy.inra.fr (P. Renault)
Biochimie 84 (2002) 10731087
www.elsevier.com/locate/biochi
2002 ditions scientiques et mdicales Elsevier SAS and Socit franaise de biochimie et biologie molculaire. All rights reserved.
PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 0 0 2 9 - 9
cal properties and increase the reliability of food making
processes, and improve product safety and quality. More-
over, LAB can be engineered to function as cell factories.
Cell metabolism can be engineered to massively produce
metabolites of interest such as food additives and aroma
compounds. They were also shown to be able to produce
proteins 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 LAB
and the human host will allow to better exploit their expected
natural potential to improve health.
2. Potential of genetic technology in LABcloning
system and engineering strategies
Genetically, LAB may have been the rst organism in
which genetic exchange was demonstrated since Pneumo-
coccus was transformed in 1928 in vivo in mice by Grifth.
The transforming principle was found to be DNA in 1944 by
O.T. Avery et al. (reported by Avery et al. [5]) and a protocol
for genetic transformation was described in 1957 by Bracco
et al. [6]. However, most genetic discoveries in bacteria have
been made in one or other of the two major model microor-
ganisms, Escherichia coli and Bacillus subtilis. A new start
in the genetics of LAB, especially those benecial to man,
came from the possibility of producing and regenerating
protoplasts [7] and nally of transforming them [8]. New
techniques such as electroporation have been developed,
allowing the transformation of most species of LAB [9]. We
should also mention alternative processes of genetic ex-
change in LAB like conjugation and transduction [10]. Most
of these mechanisms initially developed in Lactococci have
since been found to work in many other species of LAB.
The impressive increase in interest in the genetics of LAB
has led to the development of a variety of genetic systems to
analyze and modify the metabolism of these bacteria. This is
particularly evident for the species L. lactis that has become
the paradigm for LAB, but many of these tools are now
becoming available for other LAB of industrial interest. To
summarize, these systems may be classied as cloning sys-
tems, chromosome modication systems, and expression
systems [11]. We will mention here only those that are of so
called food grade and are allowed to be introduced into our
food. In general, food grade systems have to contain only
genetic elements that are as safe as the host. It is more or less
accepted that these elements must have originated from bac-
teria that already have a long history of use in food. In most
cases, these genetic elements derive fromplasmids and genes
from bacteria of the same species, to provide a self-cloning
system, facilitating their agreement for use as dened by the
novel foods procedure in the EU. In all cases, they should be
well characterized and not contain antibiotic resistance
markers, and not require the use of harmful compounds. In
addition to these safety issues and legal constraints, food
grade systems have to meet minimum requirements for sta-
bility under industrial conditions and scale of use and have to
be applicable in an efcient and cost-effective manner.
2.1. Food-grade cloning systems
A number of plasmid vector systems have been developed
using the origin of replication of natural plasmids combined
with 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 Leuconostoc
mesenteroides[15]. Integrative plasmid strategies have also
been 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 single
cross-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 dened, those that are selectable
because they confer a new phenotype, and those that restore
an impaired function. In the rst class are sugar utilization
markers such as sucrose [21] or xylose [22] genes, bacterio-
cin resistance genes such as those conferring insensitivity to
nisin [23,24] or lactacin F [25] or metal such as cadmium
[26]. For the second type of marker, any function necessary
for cell viability and that can be conditionally inactivated to
produce auxotrophic mutants can be used. Asystembased on
the inactivation of lacF, a lactose gene, has been developed in
L. lactis [27,28]. A system based on a suppressor tRNA
allowing growth in milk of a purine auxotrophic strain was
designed for industrial L. lactis strains [29,30]. Alanine
racemase mutants of L. plantarum able to grow only in the
presence of D-alanine were also constructed, and could be
complemented by the functional gene [31,32]. The present
list is not exhaustive, but shows that a number of food-grade
vectors and markers are now available in different LAB.
2.2. Targeted chromosomal modication systems
Although many vectors are now available, the systems
described in the previous section have several disadvantages:
(i) copy number of plasmids may vary, (ii) plasmids may be
lost in the absence of marker selection and (iii) plasmids may
be structurally instable. Moreover, these vectors require the
introduction DNA in addition to that of the desired gene.
Tools are also available to insert genetic constructions by
allelic replacement in the chromosome. This method has
several advantage over replicative or single cross-over inte-
gration vectors. In particular, allelic replacement allows
stable DNA insertion or genetic modications 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 modication and the corresponding
regions on the chromosome. This procedure may occur spon-
1074 P. Renault / Biochimie 84 (2002) 10731087
taneously upon transformation using the natural competence
machinery, such as those described in Streptococcus pneu-
moniae [33] and B. subtilis [34]. However, although the
genome of the sequenced LAB contain the set of genes
required for competence development ([4], personal data),
procedures to induce these systems have not yet been de-
scribed. A thermosensitive plasmid-based system has been
developed allowing gene replacement by a two-step proce-
dure (Fig. 1 ). A mutation in the gene encoding plasmid
replication protein of the natural plasmid pWV01 has been
selected, allowing maintenance of the plasmid at 30 C, but
not at 37 C [35]. This plasmid can direct homologous
integration in the L. lactis chromosome when it carries a
chromosomal DNA fragment of sufcient length (about
500 bp or more; [36]). After the two step procedure, allowing
sequential recombination in the two fragments anking the
central modication (nucleotide change, deletion or DNA
insertion), the thermosensitive plasmid vector with selective
markers can be cured by simply growing the modied strain
at 37 C for several generations to allow plasmid segregation
once replication is blocked. Lastly, this plasmid can also be
used to select food-grade mutants containing a single IS
element as new DNA fragment in the genome [37].
2.3. Expression systems
In addition to cloning systems, gene expression systems
have been developed allowing the controlled expression of
homologous or heterologous genes. Most of these systems
were developed in L. lactis, such as those based on promoters
controlled by sugar (lactose operon promoter, [38]), by salt
(gadC promoter, [39]), by temperature upshift (tec phage
promoter, [40,41]), pH decrease (P170, [42]) and phage
infection (phi31-promoter, [43]). A dose-dependant system
of induction is also available using sublethal concentrations
of nisin in L. lactis [44] and this system was extended to
some 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 production
of heterologous proteins, inducible systems are not always
easy to manage under industrial conditions, especially if a
constant level of production is required for metabolic control,
for example. In this case, a well-dened constitutive pro-
moter with the desired level of expression may be more
efcient. A system of synthetic promoters allowing the con-
stitutive and dened level of expression of downstreamgenes
has been created recently [49] and could, in principle, be
applied to any bacterial species [50].
3. Strain improvement in food technology
Since the rst starter cultures were used for fermented
dairy products, the main stream of research has been per-
formed to improve dairy starter strains, and in particular
L. lactis. 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 the construction of
improved starter strains from the most simple designs to
more complex variants.
3.1. Producing genetic variants
LAB are naturally diverse and strains belonging to the
same 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 to
combine a specic trait of one strain with another strain that
has a background well adapted to a particular process. This
could be of particular importance if only a single strain is
available for a given process, precluding the use of alterna-
tive strains in case of phage attack, for example (see below
for phage issue). There is thus a need to be able to combine
strain characters in order to produce reliably fermented food
products of high quality.
In some cases, the desired modication could be restricted
to the mutation of a single gene, a process that can occur
spontaneously, sometimes at relatively high frequencies.
Thus, lactococcal variants in lactose metabolism [51], citrate
uptake [52] and proteolytic activity [53] can easily be ob-
tained by simple screening procedures, because the genes
necessary for these metabolic pathways are encoded on seg-
regationally unstable plasmids. These variants can be se-
lected at frequencies ranging from 10
3
to 10
1
.
However, the number of traits that could be modied by
such an easy method is quite limited, and more efcient
screening strategies should be set up to select mutants arising
at frequencies of 10
6
or lower, which is approximately the
level of spontaneous mutation of a gene in the chromosome.
In some cases, screening can be facilitated by color reactions,
such as the use of X-gal to select strains devoid of
b-galactosidase activity. This screening strategy was used to
select L. bulgaricus strains unable to efciently ferment
lactose and do not acidify yogurt after fermentation has
nished [54]. Indeed, upon storage, yogurt pH may drop
belowa value of 4.0, increasing the acid and bitter taste of the
product, and thus degrading its initial organoleptic quality.
This post acidication process is mainly due to L. bulgaricus
lactose metabolism. Lactose decient strains are still able to
grow in association with S. thermophilus, the second yogurt
starter 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 for
any gene as long as its function is not essential. However, it
could be difcult to isolate the proper mutant among millions
of wild type cells, especially for spontaneous mutations that
are not stable as it is the case when it confers a slight decrease
in growth rate. Use of mutagenic compounds such as EMS or
N-methyl-N'-nitro-N-nitrosoguanidine may be used to in-
crease the rate of recovery of mutations. LDH mutants hav-
ing a mixed pattern of fermentation, and producing increased
amounts 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 used
advantageously. 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.
tis subsp. diacetylactis may be presented as a paradigm for
this issue in LAB.
Diacetyl is responsible for the butter avor in many fresh
dairy products such as butter, cream and buttermilk. How-
ever, even if its presence at low concentration is sufcient to
confer this typical aroma, diacetyl is highly labile and its loss
results in a at taste of the products. The main pathway for
diacetyl production is a two step synthesis from pyruvate.
The rst step is common to valine biosynthesis and acetoin
catabolism pathways through the reaction of two pyruvate
molecules to give a-acetolactate catalyzed by a-acetolactate
synthetase. Diacetyl is then formed by a chemical oxidation
occurring spontaneously at low rate. However, in L. lactis,
a-acetolactate is also actively decarboxylated by a-aceto-
lactate decarboxylase into acetoin, a compound that does not
confer the desired avor (Fig. 2 ). Inactivating aldB, the gene
encoding a-acetolactate decarboxylase, should thus increase
the availability of a- acetolactate for chemical oxidation. An
experimental protocol to isolate spontaneous aldB mutants
was designed with prototrophic L. lactis strains taking ad-
vantage of the misbalance in the ux of acetolactate between
valine synthesis and acetoin catabolism in the presence of
leucine [59]. As this screen can only be applied to valine
prototrophic 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 not
contain any foreign DNA. This strain accumulates high
amounts of a-acetolactate leading to stable formation of
diacetyl throughout 3 weeks of storage. Currently, this strain
is used in combination with a diacetyl reductase Leuconostoc
mutant in a starter commercialized in the USA. Although the
Danish 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 not
used in the EU [62]. Issues concerning the use of GMOs in
the EU will be discussed at the end of this paper, in the
section on risk assessment.
Gene technology allows the production of similar aldB
mutants by direct allelic replacement using an appropriate
thermosensitive vector [61]. This approach was also adopted
to obtain food-grade mutants of L. lactis resistant to phages
by 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 a
laborious screening to isolate an aldB mutant [57], central column: the aldB mutant is selected by a screen using the dual role of L. lactis acetolactate
decarboxylase in the regulation of the acetolactate pool ([58], Fig. 2) to select leucine resistant clones (Leu
R
) that do not display growth inhibition in a
chemically dened medium containing leucine but not valine and isoleucine [59]. A plasmid carrying the ilv operon should be provided to valine auxotrophic
strains [60]. Right column, two-step procedure to exchange the functional aldB allele for an inactivated copy of the gene [61]. The rst procedure leads to strains
that do not need to be labeled as GMO, while the two procedure involving plasmid transformation lead to GMOs [62].
lar approaches could also be used in other LAB species such
as S. thermophilus where the inactivation of the phosphoglu-
comutase gene enhances polysaccharide production [65] and
that of urease genes reduces delay in the acidication in
milks containing high amount of urea (Anba et Renault, in
preparation).
Lastly, specic mutant may be isolated by use of an IS
element [37]. The nal strains only contain ISS1 originating
from L. lactis at a well dened place in the chromosome, for
example in the target genes presented above. Interestingly,
ISS1 mutants affected in complex genetic and physiological
response were obtained, such as stress resistant strains af-
fected in guaA(inactivation), relA(modied activity, [66]) or
mutant in proteolysis [67,68]. In both cases, it is expected
that the expression of a number of genes is affected. In the
rst case, it is proposed that the increased resistance to stress
is due to a decrease in the intracellular GTP pool and/or
stringent response, a response involving a drastic change in
the cellular metabolism and regulation in E. coli[69] and
B. subtilis [70]. However, relatively little is known about
these 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 initially
found to regulate early-stationary-phase genes in B. subtilis
[71]. In L. lactis, this regulator was found to regulate the
most important genes involved in casein utilization. In codY
mutants, the transcription of the cell-wall protease genes, a
peptide permease operon and the major peptidase genes is
increased between 5 and 100-fold. In addition to these genes,
it is now known that CodY also regulates at least some
aminotransferases (Yvon, personal communication). Since
proteolysis and amino acid degradation have a major role in
cheese ripening [72], it is expected that codY mutants will
have some interesting properties for cheese making and the
potential interest of codY mutants is now being tested.
3.2. Constructing modied strains with genes from other
LAB
In the precedent cases, the modied bacteria could be
considered as mutants equivalent to bacteria that might al-
ready exist naturally. However, introducing newDNAencod-
ing newinformation into the bacterial cell could lead to wider
strain 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 different
species of LAB. In the rst case, the modied strains are by
self-cloning, while in the second, the notion of self-cloning
requires discussion (see the section concerning risk assess-
ment).
To obtain strains with increased proteolytic properties, the
genes encoding PepN, PepC, PepX and PepI peptidases of a
highly proteolytic L. helveticus strain [73] or PepI, PepL,
PepW, and PepG from L. delbrueckii [74] were transferred
into 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, for
example, by shortening the ripening period and allowing the
production of special cheeses (e.g. reduced-fat cheeses) with
improved 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 in
industrial productions can be highly subject to infections
with bacteriophages. This leads to the lysis of the starter and
thereby the arrest of fermentation. The products obtained
then do not have the desired quality, and may eventually be
lost. The origin of bacteriophages is still discussed, since
they may come from raw products such as milk that had
contact with environmental farm bacteria including wild
LAB, from inoculum (mostly if it is not properly produced),
from the factory itself, or from the evolution of remnant
phages present in the starters. Selection of bacteriophage
resistant 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 resistance
mechanisms (recently reviewed by Forde and Fitzgerald,
[75]). Phage resistance systems may interfere with phage
adsorption, phage DNA injection, phage replication, tran-
scription, RNA translation, protein assembly and phage
packaging. These mechanisms are often carried out by mo-
bile elements such as plasmids and transposons suggesting
that lateral transfer of these genes occurs under pressure of
phage infection. Some high level resistance plasmids were
shown to carry more than one resistance mechanisms. To
improve phage resistance, one could rationally combine
these mechanisms as a function of their target in phage
development and of the phages present in the factories. A list
of mechanisms, with emphasis on those patented has been
presented in a recent review [76].
In addition to these natural resistance mechanisms, recent
advances in the knowledge of phage biology has allowed the
generation of new weapons by targeting specic steps in
phage 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 gene
upon phage infection [78] or to massively produce antisense
mRNA against essential phage genes [79,80]. The most im-
portant drawback of these systems is their narrow range of
action. Lastly, DNA shufing, exploiting the properties of a
type I restriction enzymes could also generate new
restriction/modication 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 cheese
ripening. Indeed, whereas starter lysis is a major problem
during fermentation, late cell lysis allows the release of many
enzymes into the cheese matrix, improving in particular the
degradation of peptides. This degradation allows cheese to be
made less bitter (due to the reduction of some bitter peptide),
and contain more free amino acids (precursors of aroma).
Several approaches have been proposed to provoke con-
trolled lysis of starter bacteria, including the use of autolytic
strains, bacteriocin producing starters [82] and phages [83].
Several of these approaches are not easy to optimize, or may
even be seen as unacceptable by industry because of the use
of phages that may contaminate other processes in the fac-
tory. Engineered strains producing phage derived lysin and
holin proteins [84] or bacteriocin [85] under the control of an
inducible promoter have been constructed. Increased lysis of
the cells was obtained and cheese trials have shown that
under some conditions this lysis may allow an increased
aroma production. However, additional work is needed to
optimize the strains for industrial use. In addition to acceler-
ating cell lysis for cheese ripening, heterologous bacteriocins
could also be produced to eliminate undesirable contaminant
bacteria [86].
Lastly, we will briey mention two examples where new
functions are provided by inter LAB cloning. An amylolytic
L. plantarum silage strain with high starch-degrading ability
was developed by expressing the L. amylovorus amylase
gene. This recombinant strain may have potential as a silage
inoculant for crops such as alfalfa, in which water-soluble
carbohydrate levels are low but which contain starch as an
alternative carbohydrate source [87]. As a second example,
an L. lactis strain containing the complete eps cluster from
S. thermophilus S6 is able to produce an exopolysaccharide
of similar size to that of the native strain. However, its
composition differs suggesting that an additional chromo-
somal copy was required for its complete synthesis [88].
Similar experiments carried out with the eps cluster from
S. thermophilus S39 allowed the production of an EPS
similar to the one produced by S39 [89]. These examples
show that complete complex biosynthesis pathways can be
introduced in LAB, and in the above mentioned case, would
have application to feed production or in the improvement of
the texture of fermented food.
3.3. Modied strains with heterologous genes
A number of applications can be proposed that depend on
modifying genes or transferring genes from food LAB into
other lactic starter strains. However, some functions may not
exist in LAB, and such modication would necessitate the
transfer 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 to
produce alpha-ketoglutarate from glutamate, an amino acid
present at high levels in cheese. Indeed, during cheese ripen-
ing amino acid degradation plays a major role and its rst
step in lactococci is a transamination, which requires an
alpha-keto acid as the amino group acceptor [90]. Moreover,
the rst limiting factor for conversion of amino acids to
aroma compounds may be the level of available alpha-keto
acids. Interestingly, the GDH-producing lactococcal strain
produced a higher proportion of carboxylic acids, which are
major aroma compounds suggesting that such modied
strains could be used to avoid alpha-ketoglutarate supple-
mentation [91].
Another interesting example of genetic engineering is the
redirection of metabolism to produce high added value prod-
ucts. For example, it was proposed that LAB such as L. lactis
could be used to produce high amounts of L-alanine not
contaminated by the D-stereoisomer [92]. Alanine could be
produced 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 Bacillus
sphaericus alanine dehydrogenase was expressed in an
L-LDH-decient lactococcal strain. The constructed strain
produced alanine as the sole end product. Finally, stereospe-
cic production (>99%) of L-alanine could be achieved by
inactivating the host-gene encoding alanine racemase.
Although of great potential interest, the use of LABmodi-
ed with exogeneous genes may be limited, at least in the
near future, by the suspicion consumers may have concern-
ing the use of such genetically modied 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 more
positive.
4. Potential application of LAB to improve health
The consumption of specic LAB, mainly members of
Lactobacillus, has been proposed to be benecial for human
health, by the prevention of gastrointestinal tract infections,
by immune stimulation, and by the balancing of intestinal
microora. These potential health promoting bacteria are
often 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 probiotic
characteristics often remain controversial and further
progress concerning the molecular basis of probiotic traits
will be a prerequisite for the rational development of further
applications [93,94]. Interestingly, other approaches to the
use of LABfor promoting health was proposed by modifying
strains that initially had no known probiotic effect. In this
case, the term probiotic should probably be avoided since
these bacteria were constructed for direct medical applica-
tion and not for distribution to consumers (in food or by other
means) without medical control. We will focus our attention
on these applications in this section.
Progress in medical science allows the design of strategies
targeted to cure disease, including drug delivery, the supply
of molecules compensating defects or the stimulation of
human functions such as the immune system. Some of these
strategies make use of molecules that could be synthesized
by bacteria, including LAB. Most work in this domain has
been devoted to the development of new vaccine strategies,
but has also concerned substitution therapy to correct en-
zyme defects in the digestive tract [95].
4.1. Vaccine development and modulating the immune
system
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 to
deliver protective antigens at mucosal surfaces is to use live
bacterial vectors. Over the last two decades, the use of re-
combinant bacteria as carrier system to deliver antigens to
the 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]. Anumber of other bacteria such
as Listeria, Vibrio, Bordetella, Mycobacterium have also
been proposed, although the major concern in the use of
attenuated pathogens is that they may still be virulent in the
elderly and in very young children. Food LAB usage would
overcome this problem since these bacteria have a long
history 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 commensal
Streptococcus gordonii, mainly within the framework of
several European programs (reviewed by Mercenier et al.
[97]). A selection of various studies is presented inTable 1 .
Among the rst and most extensively studied antigens is
fragment C of tetanus toxin, which has been massively pro-
duced in L. lactis and shown to protect mice immunized
subcutaneously 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 administration
and that other LAB could also be used for this purpose [101].
Vaginal immunization could be induced by the use of S. gor-
donii[105107]. 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 inammatory bowel diseases [112].
Studies have been initiated to better understand allergy and
eventually modulate responses to food allergens [115].
Lastly, new strategies have been proposed against experi-
mental candidiasis by the expression of an anti-idiotype in
S. gordonii. This human commensal bacterium was engi-
neered to locally release a microbiocidal single-chain anti-
body. The bacteria stably colonized rat vagina and allowed
the treatment of experimental vaginitis caused by Candida
albicans [118,124]. The same team also expressed another
anti-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 ght
against S. mutans involved in dental caries [125].
Although these approaches are promising, many ques-
tions remain unanswered. Among these are the ecology of the
carrier LAB that could persist in the host or be rapidly lost.
Biologically contained strains could be the goal of future
improvements [97]. Methods to tag bacteria or to study their
physiology upon administration are now available for this
Table 1
Examples 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 immunodeciency
virus gp41 protein)
Vaginal immunization for HIV [104]
S. godonii, L. casei V3 domain of the gpl20 of human immunodeciency
virus type 1 (HIV-1)
Vaginal immunization for HIV [105107]
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 inammatory 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 insufciency [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 capsular
polysaccharide of group B streptococci
Passive protection of neonatal pups from group B
streptococci 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]
purpose [126,127]. Moreover, it has been suggested that
antigen presentation may be of crucial importance in immu-
nization, and that certain constructs could induce immuno-
tolerance rather than immunization [128]. Different tools are
now available to present antigens in a different way and such
issues could be tested in model systems [98,99,103,
113,122,129]. Possible limitations due to secretion machin-
ery bottlenecks are also under investigation [130133].
4.2. Compensation for metabolic defects
The potential of LAB to correct metabolic defects has
been relatively less studied. However, it has been known for a
long time that fermentation by LAB reduces lactose intoler-
ance [134]. Although it is controversial, it has been suggested
that bacterial lactase could naturally supplement the human
enzyme in cases of deciency. The idea that LAB could
compensate for enzyme deciency is thus not new. Recently,
lipase from S. hyicus was produced massively intracellularly
in 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 and
massively lyse in the jejunum where the lipase should be
delivered [126]. Oral treatment with L. lactis expressing this
lipase was carried out in a pig model where pancreatic insuf-
ciency was induced by ligation of the pancreatic duct. The
coefcient of fat absorption was signicantly higher after
consumption of lipase-expressing L. lactis than after con-
sumption of the control strain showing that this strategy
could in principle be applied to compensate for enzyme
deciency [133].
5. Other applications
In addition to food and health applications, genetically
modied LABhave been used as improved biosensors for the
detection of biocides in milk. Initially, a commercial product
made use of a S. thermophilus strain particularly sensitive to
antibiotics to detect possible contamination of milk that
might perturb fermentation. This test requires a few hours to
assess whether the metabolic activity of the strain is inhib-
ited. To shorten it, luciferase genes were introduced in this
strain and their expression optimized [135]. Finally, using
reduced light production in highly bioluminescent S. thermo-
philus, test times could be signicantly shortened compared
to the previous commercial test utilizing the related non-
bioluminescent strain. This GMO, presenting an improved
test kit to detect antibiotic residues in milk (Valio Oy), is the
only LAB which was approved under the directive
90/220/EEC since December 1997. It should be mentioned
that this bacterium is not present in food products and is
destroyed after the test has been realized on a small sample.
6. Risk assessment
Fermentation-based bioprocesses rely extensively on
strain improvement for commercialization. Until now, strain
improvement was mainly based on the selection of new
natural 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 modied LAB are
commercialized as yet, and some aspects underlying this
issue will be discussed here. Community legislation on
GMOs has been in place since the early 1990s, but this
regulatory framework has been further extended and rened.
It was designed to protect the health of EU citizens and the
environment at the same time as the creation of a unied
market.
6.1. EU legislation and risk assessment procedure
To date, Directive 90/220/EEC is the main legislation
authorizing experimental and commercial release of GMOs
in the Community. An updated Directive 2001/18/EC on the
deliberate release of GMOs should apply on 17 October
2002. Directive 90/220/EEC put in place a step-by-step ap-
proval process on a case-by-case assessment of the risks to
human health and the environment before any GMO or prod-
uct consisting of or containing GMOs can be released into
the environment or placed on the market. Products derived
from GMOs are covered by the Regulation on Novel Foods
and Novel Food Ingredients (258/97). Lastly, Directive
90/219/EEC amended by directive 98/81/EC regulates the
contained use of GMMs for research and industrial purposes.
The objective of risk assessment is to identify and evaluate
potential adverse effects of GMOs. These effects could be
either direct or indirect, immediate or future. The cumulative
and long term effects on human health and the environment
have also to be taken into account. The risk assessment looks
specically at howthe GMproduct was developed and exam-
ines the risks associated with the gene products in the product
(for example toxic or allergenic proteins), but also after a
possible gene-transfer (for example antibiotic resistance
genes). The determination of the overall risk of the GMOs
requires the identication of any characteristics of the GMOs
which might cause adverse effects, the evaluation of their
potential consequences and their possible occurrence. From
these 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 to
the use of GM plants and the most recurrent issue raised by
opponents is our lack of knowledge of the effect of potential
gene transfer to the environment. However, test experiments
set up by governmental institutes to measure gene ow are
also 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 that
GMOs are an industrial advance that prot only a small
fraction of the population. Although most of these issues are
not relevant for microbial GMOs, the climate of mistrust
covers all GMOs, except perhaps those targeted to medical
uses. These social issues are far beyond the scope of this
review, and to summarize, it is possible to obtain approval for
the use of GMOs in the EU, but the subsequent use of these
GMOs in the open market is hampered by the mistrust of
consumers and a strict labeling legislation that allows the
consumer to select products as a function of their content in
GMOs (90/220/EEC and Council regulation 1139/98) or
derived 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 use
of GMOs in the EU, we will not speculate about the evolution
of consumer attitudes in this section. We will rather discuss
what could/should be or not be considered as a GMO and
labeled as such. Indeed, in the EU GMOs (animals, plants
and microorganisms) are dened as organisms in which the
genetic material has been altered in a way that does not occur
naturally by mutation, mating or natural recombination. Ge-
netic engineering technology allows selected individual
genes to be transferred from one organism into another, even
between non-related species. Is this denition relevant in the
light of current scientic knowledge including, in particular,
genomics? If we consider that safety issues should predomi-
nate in the GMO debate, what would be the safest procedure
for strain improvement?
First, it should be underlined that the GMO denition is
not applied similarly in all EU countries: indeed, is the nal
construction only relevant or should the way in which the
modication was done also be considered? If we take as an
example the diacetyl overproducing strains, the same muta-
tion inactivating the aldB gene could in principle be obtained
in different ways, i.e. spontaneous mutation, induced mu-
tagenesis, or genetic engineering (see 3.1 and Fig. 3). In
Denmark a spontaneous mutant isolated after a transient step
in which the strain contained foreign DNA has been consid-
ered as a GMO, although this strain should be similar to and
possibly even less modied than one obtained by chemical
mutagenesis [62]. Also, a mutant obtained by allelic replace-
ment of aldB by a modied copy of the gene, even if the
mutation is a deletion similar to a natural event would be
considered as a GMO. This is due to the fact that in addition
to the nal product (that the US regulation takes only into
account), the EU regulation considers to be relevant the
way the modication was performed. In terms of safety
evaluation, 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 UVor chemicals
often produces secondary mutations due to the lack of target-
ing of the method. On the other hand, genetically driven
mutagenesis is considered to allow targeted modications
and should thus avoid additional mutations. However, the
formal demonstration of the directness of the latter technique
has not been implemented, especially because until recently,
side effects could be determined only with difculty due to
the lack of appropriate technology. An EU founded program,
Express-Fingerprints (QLK3-2001-01473) is presently
testing the potential side effects due to the technology in
L. lactis. For this purpose, aldB and pip mutants obtained by
chemical mutagenesis and gene technology will be compared
by two global analytical methods: 2Dgel electrophoresis and
transcription proling 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 450
cytoplasmic proteins and of the transcription of over 2100
L. lactis IL1403 genes larger than 89 bp. In addition to
pinpoint possible side effects, Express-Fingerprint work
should also point out the deregulation due to the wanted
change itself. Global expression proles will thus allow(i) to
determine 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 from
more distantly related species could be developed. In that
case, at least three issues should be addressed: (i) is the
product of the new gene(s) safe, (ii) does the new gene(s)
induce undesirable functions in the new host and (iii) is there
any danger in case of transfer of the new gene? The rst and
the last issues should be examined case by case, while the
second could be assessed by the same strategy as that pro-
posed by Express-Fingerprints. Several strains of L. lactis
expressing heterologous proteins will be tested in order to
evaluate 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 inuence
safety. Indeed, whereas it could be reasonably considered
that many mutants (punctual, insertion of IS, deletion) are
substantially equivalent to strains naturally occurring (for
example, if a specic mutation is expected to occur at a very
low rate, i.e. 10
9
per generation, more than 100 such cells
will be present in a yogurt containing 10
9
viable cells per g),
it could be asked what additional genes could be added
without giving the GMO label? As previously shown, many
of the genes transferred to improve the technology or quality
of a given starter strain are shed from a pool of genes
present in the same or less closely related species. We have
now evidence of many similar transfers in nature; further-
more, gene shufing occurs on the chromosome
[81,136,137], on plasmids [138,139], on transposons [140]
or on phage related elements [141,142]. Establishing the
limit of the natural gene pool might not be a trivial issue.
Unfortunately, transfers including undesirable genes may
occur naturally between bacteria from different species
[143]. New genome plasticity data show that strains in the
same species may signicantly differ [144,145]. Most of
these studies were done on pathogenic bacteria, but major
evolutionary mechanisms might be similar in pathogenic,
environmental and food bacteria. Gene transfer has been
shown between pathogenic and commensal bacteria [146].
Vehicles for these transfers include phages that have been
shown to be related between pathogens and food bacteria
[147]. Phages are also known to be vehicles for pathogenicity
islands in the former bacteria [148,149]. Therefore, natural
transfer of genes between pathogenic and natural isolates of
food species cannot be excluded. Importantly, food bacteria
are often derivatives of environmental and commensal bacte-
ria [150], in particular probiotic strains that are isolated from
intestinal ora [151,152]. Thus, how reliable is the assump-
tion that a function isolated from a strain belonging to a
bacterial species that has a long history of safe use will be
safe in another background?
Assessment by global analyses such as done in the
Express-Fingerprints program on a set of natural strains will
only underline the variation in gene expression of genes and
functions already characterized during the annotation of the
genome and used to design the DNA-microarrays. Any new
DNA fragment, and its expression products will not be de-
tected (except possibly by proteome analysis) although it
could encode new traits. It might thus appear that GM micro-
organisms are much better characterized than newstrains of a
genuine food species. However, one could also expect that
new high-throughput genomic technologies will allow the
rapid characterization of new DNA elements present in new
strains. These technology could eventually also be used to
control 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 shufing are now
emerging [153]. Strain produced by these methods may not
be considered as a GMOalthough the derived bacteria will be
less well characterized than tailored GMOs. Genome shuf-
ing was successfully applied to improve the acid tolerance
of a poorly characterized industrial strain of Lactobacillus
[154]. In the rst step, classical strain-improvement methods
were used to generate populations with subtle improvements
in pH tolerance. In the second step, these modications were
shufed by recursive pool-wise protoplast fusion. New
shufed lactobacilli that grow at lower pH or produced more
lactic acid than does the wild-type strain were identied. The
authors conclude that genome shufing seems broadly useful
for the rapid evolution of tolerance and other complex phe-
notypes in industrial microorganisms. It should be added that
genome shufing is a naturally occurring process using the
natural cellular machinery, such as competence, as shown in
pathogenic Streptococcus species. Genome analysis on food
bacteria shows that the full set of genes necessary for this
process is present in the completed genomes of L. lactis [4]
and S. thermophilus, a food bacteria closely related to the
human commensal S. salivarius (Bolotine and Renault, per-
sonal data). The concept that GMO are dened as organisms
in which the genetic material has been altered in a way that
does not occur naturally by mating or natural recombination
may be seen as obsolete in the near future. Moreover, study
of genome evolution will probably provide us with numerous
examples of organisms in which the genetic material has
been altered in ways far more complex than man will be able
to mimic.
Acknowledgements
Part of the work for this review was done within the
framework of the Express-Fingerprints program (QLK3-
2001-01473) of the European Commission. We thank Jan
Kok 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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