Genetically Engineered Lactobacilli for Technological and Functional Food Applications

Lactic acid bacteria (LAB) are Gram-positive microorganisms that produce lactic acid as a major product of their metabolism. Among them the genus Lactobacillus comprises a large heterogeneous group of low G+C DNA content, anaerobic and nonsporulating bacteria, that includes species widely used in the food industry. They play key roles in fermented dairy, meats and vegetables products. Due to their claimed health-promoting properties certain lactobacilli species are used as probiotics and they are commonly applied to dairy and functional foods products. Lactobacilli have a relatively simple fermentative metabolism focused to rapidly convert carbohydrates into lactic acid, and exhibited a limited biosynthetic capacity. In addition, several tools and strategies to manipulate them genetically are available. All those characteristics make lactobacilli specially suited for genetic engineering aimed to increase existing compounds or to produce novel metabolites of interest for the food industry. Regarding probiotic lactobacilli, through genetic manipulation, the health attributes of probiotic strains can be enhanced or new probiotic activities can be developed and additionally, an understanding of the underlying molecular mechanisms can be obtained. Here, we review metabolic engineering strategies in lactobacilli that have successfully been used to efficiently reroute sugar metabolism to compounds such as L-lactic acid, aroma compounds (acetoin, diacetyl), low-calorie sugars (mannitol, sorbitol) and exopolysaccharides. We will also describe strains of probiotic lactobacilli that have been developed to exploit their adherence and immunomodulatory properties, and to delivery proteins at the intestinal mucosa.


Introduction
Lactic acid bacteria (LAB) are Gram-positive microorganisms that produce lactic acid as a major product of their metabolism.Among them the genus Lactobacillus comprises a large heterogeneous group of low G+C DNA content, anaerobic and nonsporulating bacteria, that includes species widely used in the food industry.They play key roles in fermented dairy, meats and vegetables products.Due to their claimed health-promoting properties certain lactobacilli species are used as probiotics and they are commonly applied to dairy and functional foods products.Lactobacilli have a relatively simple fermentative metabolism focused to rapidly convert carbohydrates into lactic acid, and exhibited a limited biosynthetic capacity.In addition, several tools and strategies to manipulate them genetically are available.All those characteristics make lactobacilli specially suited for genetic engineering aimed to increase existing compounds or to produce novel metabolites of interest for the food industry.Regarding probiotic lactobacilli, through genetic manipulation, the health attributes of probiotic strains can be enhanced or new probiotic activities can be developed and additionally, an understanding of the underlying molecular mechanisms can be obtained.Here, we review metabolic engineering strategies in lactobacilli that have successfully been used to efficiently reroute sugar metabolism to compounds such as L-lactic acid, aroma compounds (acetoin, diacetyl), low-calorie sugars (mannitol, sorbitol) and exopolysaccharides.We will also describe strains of probiotic lactobacilli that have been developed to exploit their adherence and immunomodulatory properties, and to delivery proteins at the intestinal mucosa.

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Food Industrial Processes -Methods and Equipment 146 1.2 Functional properties of lactobacilli Bacterial populations in the gut of vertebrates have evolved through millions of years to render interdependent functions.They have been studied for long time using classical culturing techniques and recently through molecular approaches and it soon became evident that in humans, the gut's microbioma is formed by numerous bacterial species whose proportions change between individuals (Eckburg et al., 2005).The most abundant genera are Bacteroides, Faecalibacterium or Bifidobacterium, however, although lactobacilli are not as abundant, they have been proved to play a remarkable role sustaining the global population balance and interact at different levels with the intestinal mucosa.In this environment some strains exerted beneficial health effects and they are considered probiotics.These are defined as live microorganisms that, when administered in adequate amounts, confer a beneficial effect on the health of the host (FAO/ WHO, 2001).In addition to probiotics, functional food ingredients also include prebiotics, which are define as selectively fermented ingredients that allow specific changes in the composition and/or activity of the gastrointestinal microbiota that confer benefits upon host wellbeing and health (Roberfroid, 2007).Several beneficial effects of lactobacilli on human host have been reported.Strains of Lactobacillus rhamnosus, Lactobacillus acidophilus and L. bulgaricus alone or in combination are effectives in reduce the risk of acute infectious diarrhoea and prevent antibiotic-associated diarrhoea (Sazawal et al., 2006).A mixture of probiotics including lactobacilli seems effective in the maintenance of remission of intestinal bowel diseases such as chronic pouchitis and ulcerative colitis, and to decrease symptoms in patient with irritable bowel syndrome (Haller et al., 2010).A synbiotic food composed of the prebiotic oligofructose-enriched inulin, L. rhamnosus GG and Bifidobacterium lactic Bb12 was able to alter favourably several colorectal cancer markers in patients with cancer of colon (Rafter et al., 2007).Besides to gastrointestinal disorders, lactobacilli have also showed positive effects in other pathologies, such as in the treatment and prevention of bacterial vaginosis (Falagas et al., 2007), the prevention of atopic eczema (Tang et al., 2010) and prevention of dental caries (Stamatova & Meurman, 2009).The health promoting effects of probiotic bacteria are mediated mainly by three mechanisms, (i) microbe-microbe interactions; (ii) beneficial interactions with gut epithelium and (iii) immunomodulatory interactions (Lebeer et al., 2008).Regarding the first mechanism, probiotics can have a beneficial effect on the host by modifying the microbiota trough competition and cooperation for nutrients, production of antimicrobial compounds (lactic acid, bacteriocins, H 2 O 2 ), competition with pathogens for attachment sites to the host mucosal surface and by bacterial cell-host cell communication.With respect to the beneficial interactions of probiotics with gut epithelium, this constitutes the main target tissue of probiotic action, and Lactobacillus molecules can modify it by affecting the metabolic and barrier functions of the epithelial cells.The preservation of the epithelial barrier by probiotic lactobacilli has been attributed to induction of mucin secretion, enhancement of tight-junction functioning, upregulation of cytoprotective heatshock proteins and prevention of apoptosis of ephitelial cells.The gut mucosal surface is continuously exposed to pathogens, beneficial mutualistic and commensal bacteria, and it is armoured with the largest part of the immune system in the organism, with lymphocytes scattered in the lamina propria or in organized gut-associated lymphoid tissues (GALT) such as the Peyer's patches of the small intestine and mesenteric lymph nodes (MLNs).Those immune cells can discriminate pathogens from harmless antigens, preventing an inappropriate immune response to harmless bacteria, thorough regulatory mechanisms known as "oral tolerance", which is an still incompletely understood active nonresponse to dietary and commensal enteric bacteria or food derived antigens administered orally, also related to the maintenance of homeostasis in the gut (Murphy et al., 2007).The subepithelial dendritic cells (DCs), B cells and T cells, in the lamina propria and GALT express a wide range of pattern-recognition receptors (PRRs), surface Toll like receptors (TLRs) and intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), to acquire antigens from the intestinal lumen.Then, secreted cytokines and chemokines from DCs will determine tolerance and active immune responses against a particular antigen, and whether lymphocyte differentiation, innate, adaptive or allergy immune responses will be displayed (Hart et al., 2004).Also intestinal epithelial cells (IECs) at the mucosal surface express PRRs and can also secrete cytokines and regulatory molecules, therefore, they participate actively in the discrimination of both pathogenic and commensal bacteria (Artis, 2008).In the gut, certain Lactobacillus strains have been proved to play a remarkable role sustaining the global population balance through their ability to synthesize antagonistic compounds that restrain the proliferation of a number of pathogens.However, their mutualistic behaviour with the host involves different levels of interaction with the intestinal mucosa, resulting in an anti-inflammatory effect and restoration of the mucosal homeostasis (Haller et al., 2010).The proinflammatory cytokine profiles occasionally induced by some lactobacilli in model systems (Dong et al., 2010) be related to the moderate degree of inflammation (physiological inflammation) elicited by some commensals and moderate pathogens, and it has been conceived as a beneficial feature that creates a state of awareness for a rapid immune defence response against possible infective aggressions, while preserving homeostasis (Sansonetti & Medzhitov, 2009).

Tools and strategies for genetic manipulation of lactobacilli 1.3.1 Gene cloning vectors, genetic markers and promoters
Several constitutive or inducible gene expression systems have been developed for lactobacilli (Fang & O'Toole, 2009).The vectors have different parameters such as copynumber and host-range, they are derivatives of the rolling-circle plasmids pWVO1 or pSH71 from L. lactis or the theta-type plasmids pAM1 from Enterococcus faecalis (Perez-Arellano et al., 2001) and pRV500 from Lactobacillus sakei (Crutz-Le Coq & Zagorec, 2008).Vectors for controlled expression are mainly based on genes and promoters involved in bacteriocin production, sugar utilization genes and stress resistance.In addition, following the production of proteins by LAB using a specific expression vector they should be properly folded, targeted and sometimes recovered.Several vectors included secretion cassettes, such as those based on the secretion signal of the lactococcal Usp45 protein (Schotte et al., 2000), the expression and secretion signals of S-layer proteins (Savijoki et al., 1997), the PrtP signal sequence (Kajikawa et al., 2010) or the M6 carboxy-terminal domain to anchor proteins to the cell wall (Reveneau et al., 2002).The nisin-controlled expression (NICE) system, based on the autoregulation mechanism of the bacteriocin nisin, is a very effective expression vector for production of heterologous proteins in LAB (Mierau & Kleerebezem, 2005).The NICE system contains the promoter nisA conducting gene expression under the control of the transcriptional regulator NisR, which is modulated by phosphorylation due to the histidine-protein kinase NisK immersed in the cytoplasmic membrane.The expression of the genes placed behind the P nisA is induced by the addition of subinhibitory concentrations of nisin into the culture medium, in such a way that increasing amounts of nisin resulted in a linear dose-response curve.Optimization www.intechopen.comFood Industrial Processes -Methods and Equipment 148 of the NICE system includes the incorporation in the vectors of the nisin immunity gene nisI, which resulted in better tolerance of the cells to high amounts of the inducer nisin (Oddone et al., 2009).The NICE system was created for expression of genes in L. lactis but it has been adapted to other low-GC Gram-positive bacteria including Lactobacillus helveticus (Kleerebezem et al., 1997), L. plantarum (Pavan et al., 2000), Lactobacillus brevis (Avall-Jaaskelainen et al., 2002), L. casei (Hazebrouck et al., 2007), L. salivarius (Sheehan et al., 2006) and L. reuteri (Wu et al., 2006).In these species different strategies have been used to express the nisRK genes: on a different plasmid in relation to the nisA promoter with the target gene, both on the same plasmid or with the nisRK genes inserted into the chromosome.Similar to the NICE system, in L. plantarum (Mathiesen et al., 2004) and L. sakei (Axelsson et al., 2003) vectors have been developed using a pheromone-regulated bacteriocin promoter and the regulatory system of sakacin A production, respectively.The pSIP vector series, based on the genes and promoters involved in sakacin A and P, used erythromycin as selection marker (Sorvig et al., 2005).In order to developed a potential food-grade expression system the erythromycin gene in the pSIP vectors has been replaced by the alr gene, which encodes the alanine racemase enzyme that is essential for cell wall biosynthesis (Nguyen et al., 2011).In L. casei an integrative vector, pIlac, has been constructed that allowed stable gene insertion in the chromosomal lactose operon.The vector is based on the nonreplicative plasmid pRV300 and it contains the 3' end of lacG and the complete lacF gene (Gosalbes et al., 2000).Both vectors, pSIP and pIlac, are based on the complementation host/marker system, a gene in the host is mutated or deleted, and a wild copy is inserted into the vector.Other potential food-grade vectors are based on a selection marker that confers a new ability to the host strain.In this sense a vector has been recently developed that contains a bile salt hydrolase gene from L. plantarum and which allows the host to grow in media containing bile salts (Yin et al., 2011).Bioluminiscence markers have also been used in lactobacilli and they are based on genes encoding enzymes that produce light as lux, which encodes bacterial luciferase, and gfp that encodes green fluorescence protein (Chang et al., 2003;Perez-Arellano & Perez-Martinez, 2003).

DNA mutagenesis systems: integration and insertion systems, and random mutagenesis systems
There are two principal methods to generate mutations in lactobacilli: (i) integration, which is a rec-dependent recombination of cloned DNA with an homologous locus; (ii) recindependent, which involves transposons and insertion elements (Fang & O'Toole, 2009).The integration procedures mostly used in LAB are based on vectors able to integrate by homologous recombination with known chromosomal genes, causing their disruption by inserting foreign genes.The integrative vectors developed for lactobacilli are either based in temperature-sensitive replicons such as pG+host, pIP501, pTNI and pGID or non-replicative plasmid such as pUC18/19 and pBlueScript SK -.As well, a two plasmids system have also been used to direct integration into Lactobacillus chomosomes via homologous recombination (Russell & Klaenhammer, 2001).This system utilizes pOWV01-derived vectors from which the repA gene has been removed.The repA is supplied in trans in a temperature-sensitive helper vector.A subsequent temperature shift selects for loss of the helper plasmid and integration of the pOWV01-derived vector.In addition, there are other mutagenesis systems as that of the Cre-lox-based system used in L. plantarum (Lambert et al., 2007) and site-specific integrative vectors based on prophage fragments (Martin et al., 2000).Other important genetic tool used to study chromosomal genes and their regulation in lactobacilli is random transposon mutagenesis.The insertional sequence ISS1, combined with the thermosensitive pG+ replicon, was used to inactive genes involved in the regulation of phenolic acid metabolism in L. plantarum (Gury et al., 2004) and several genes in L. salivarius (Mason et al., 2005).Tn5 transposome system was also efficiently used to generate a library of transposon insertion mutants in L. casei (Ito et al., 2010).As well, factors affecting the reduction of serum cholesterol by L. acidophillus were identified by random transposon mutagenesis (Lee et al., 2010).

Lactic acid production
Lactic acid produced by many LAB is a racemic mixture of L-lactate and D-lactate isomers.D-lactate is not metabolized by humans, then L-lactate is the most important isomer for food biotechnological applications, and also for pharmaceutical and biopolymers industries.Many efforts have been made to construct LAB strains affected in one or several of the identified ldh genes, as they can be used in the production through fermentation of nonracemic, optically active lactic acid.In L. casei BL23, a strain that has been widely used for genetic, physiological and biochemical studies, five genes encoding proteins with LDH activity have been described (Rico et al., 2008).Mutant strains for those genes demonstrated the involvement of each ldh gene in L-and D-lactate formation in this bacterium.Gene ldh1 codes for an L-LDH responsible for the main synthesis of L-lactate, whilst hicD encodes a Dhydroxyisocaproate dehydrogenase which renders D-lactate.However, an L. casei BL23 ldh1 mutant still produced substantial amounts of L-lactate and an increase in the production of D-lactate was observed (Viana et al., 2005).D-lactate was probably synthesized via the activity of HicD, since it was abolished in a ldh1 hicD double mutant.ldh2, ldh3 or ldh4 single mutations or combined with an ldh1 deletion (ldh1 ldh2, ldh1 ldh3,ldh1 ldh4) had a low impact on L-lactate synthesis showing that ldh2, ldh3 and ldh4 genes play a minor role in lactate synthesis (Rico et al., 2008).Comparable behaviour has been reported for many LAB where ldhs have been deleted.In this sense, mutation of the genes encoding L-and D-LDHs from L. plantarum, an organism which produces a mixture of 50% D-and 50% L-lactate, never resulted in a complete lack of lactate production (Ferain et al., 1996).An ldhL mutation in L. sakei, a lactic acid bacterium which lacks D-lactate dehydrogenase activity, rendered a strain with strongly reduced L-and D-lactate production (the D isomer was a consequence of the presence of a racemase activity able to transform L-into D-lactate), but small amounts of lactate were still produced (Malleret et al., 1998).Recombinant strategies have also been used in Lactobacillus strains to produce lactic acid from sugars others than glucose and from biomass such as starch and cellulose.In an L. plantarum ldhL1 strain, that only produced Dlactate from glucose, the phosphoketolase gene was substituted by a transketolase gene from L. lactis, and the resulting L. plantarum ldhL1-xpk1::tkt strain produced 38.6 g/l of Dlactate from 50 g/l of arabinose (Okano et al., 2009).The production of D-lactate from xylose was also achieved in L. plantarum by disrupting a phosphoketolase 2 gene in the L. plantarum ldhL1-xpk1::tkt strain and transforming it with a plasmid that contains the genes xylAB.The L. plantarum ldhL1 strain was transformed with plasmids expressing amylolytic or cellulolytic enzymes, and the resulted strains were able to produce D-lactate from raw corn starch or cellulosic compounds, respectively (Okano et al., 2010).
In addition to rational methods of metabolic engineering, lactic acid production has also been enhanced by a combination of classical strain improvement methods (nitrosoguanidine and ultraviolet mutagenesis) with whole-genome shuffling by recursive protoplast fusion.In this way, shuffled strains derived from an industrial strain of Lactobacillus have been isolated, and they produce threefold more lactic acid than the wild type at pH 4.0 (Patnaik et al., 2002).Shuffled L. rhamnosus strains with improved tolerance to glucose and enhanced Llactate production has also been obtained (Yasuda et al., 2008).In the same way, a fusant derived from Lactobacillus delbueckii able of growing at low pH and utilizing starch from cassava bagasse was obtained and it produced large amounts of L-lactic (John et al., 2008).

Diacetyl and acetoin production
Diacetyl and acetoin are important compounds of buttery flavor in fermented foods and are used as additives in the food industry.Both compounds are derived from pyruvate, which is converted to -acetolactate by the action of -acetolactate synthase or acetohydroxyacid synthase.Then, acetoin is formed by the activity of -acetolactate decarboxylase on acetolactate and diacetyl results from a non-enzymatic oxidative decarboxilation of acetolactate (Figure 2).Most metabolic engineering approaches to produce diacetyl/acetoin by fermentation have been developed in the model LAB L. lactis, in which strains that divert an important part of pyruvate flux towards the production of -acetolactate have been constructed (Hugenholtz et al., 2000;Lopez de Felipe et al., 1998).ilvBN genes, encoding acetohydroxyacid synthase from L. lactis, have been expressed from the lactose operon in L. casei, an organism which shows marginal production of diacetyl/acetoin, resulting in increased diacetyl formation (Gosalbes et al., 2000).In addition, to enhance diacetyl/acetoin production, the amount of pyruvate available for IlvBN was increased by blocking pyruvate alternative pathways in L. casei.Thus, the L. casei strain that expresses the ilvBN genes was mutated in the ldh gene and in pdhC, encoding the E2 subunit of the pyruvate dehydrogenase enzyme.The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose fermentation in whey permeate (1400 mg/l at pH 5.5) (Nadal et al., 2009).

Mannitol and sorbitol production
Sugar alcohols are hydrogenated carbohydrates widely used in the food industry as sugar replacers.Mannitol and sorbitol are used as food additives due to their sweetening effect (about half as sweet as sucrose) and low calorie content.They are also used in the food and pharmaceutical industries due to their technological properties, such as texturing agents, humectants, softeners and color stabilizers.In nature, mannitol is found in some plants, algae and mushrooms, and sorbitol is found in many fruits and vegetables.Those polyols are also produced by fungi, yeast and bacteria, where they play several roles in carbon storage and protection during osmotic and oxidative stresses.Industrial production of most sugar alcohols is performed by catalytic reduction of sugars with hydrogen gas and nickel at high temperature and pressure, for which highly pure sugar substrates and costlychromatographic purification steps are required.Regardless the limitations of this chemical method, it is until now the only process able to assume the high market demand of sorbitol and mannitol, estimated to be thousands of tons per year.However, processes using bacteria and yeasts have demonstrated that biotechnological production may represent an efficient and cost-effective alternative to the chemical production.The production of polyols by using genetically engineered LAB has been recently reviewed (Monedero et al., 2010).Mannitol is a natural fermentation product in heterofermentative LAB, in which the NADH generated during sugar metabolism is regenerated by the production of lactate and ethanol.However, in the presence of fructose, a mannitol dehydrogenase activity (MDH) can account for NADH recycling with the concomitant production of mannitol.Homofermentative LAB, which use the glycolytic pathway for sugars fermentation and lack MDH, are also able to produce mannitol under special circumstances (Figure 2).Mutants of L. plantarum and L. casei impaired in NADH regeneration by the lactate dehydrogenase were able to produce small amounts of mannitol from glucose due to a mannitol-1-P dehydrogenase (M1PDH) activity on fructose-6P (Ferain et al., 1996;Viana et al., 2005).M1PDH can recycle NADH rendering mannitol-1P that can be dephosphorylated to mannitol and excreted from the bacterial cell.M1PDH activity is generally low, because its gene (mtlD) is only induced by the presence of mannitol.Furthermore, mannitol is also a common carbon and energy source that can be fermented.Therefore, subsequent re-uptake and metabolism of the produced mannitol should be avoided.In bacteria, mannitol is usually taken up by a mannitol-specific phosphoenolpyruvate: sugar phosphotransferase system (PTS Mtl ) which catalizes the simultaneous mannitol uptake and phosphorylation to mannitol-1P.L. lactis ldh mutants have been constructed which were deleted in mtlA and mtlF, encoding the EIICB Mtl and EIIA Mtl components of the PTS Mtl , respectively, involved in mannitol uptake (Gaspar et al., 2004).This resulted in strains unable to utilize mannitol which converted 33% of the fermented glucose into mannitol.In another approach, the M1PDH encoding gene from L. plantarum and a gene encoding a mannitol-1P phosphatase from the protozoan parasite Eimeria tenella were overexpressed by using the NICE system in an L. lactis ldh mutant (Wisselink et al., 2005).This strategy avoided the main bottleneck in mannitol production: most mannitol was accumulated inside the cell as mannitol-1P, which could reach concentrations up to 76 mM in high density non-growing cells of an L. lactis ldh mutant (Neves et al., 2000).In this new strain 50% of the glucose was converted to mannitol (maximum theoretical yield of 67%).Other alternatives comprise the expression of MDH genes from heterofermenters.The mdh gene from L. brevis was expressed in a L. plantarum strain deficient in both ldhL and ldhD genes, and resulted in an increase in mannitol synthesis from glucose (Liu et al., 2005).The sorbitol (gut) operon of L. casei contained the genes gutCBA, encoding the EII component of the sorbitol-specific PTS involved in sorbitol transport and phosphorylation, two regulatory genes, gutR and gutM, and the gene gutF, encoding a sorbitol-6P dehydrogenase (S6PDH) (Alcantara et al., 2008).A recombinant strain of L. casei with the gutF gene integrated in the chromosome at the lactose operon produces sorbitol from fructose-6P by reversing the sorbitol catabolic pathway (Nissen et al., 2005) (Figure 2).Resting cells of this strain synthesized small amounts of sorbitol from glucose, with a conversion rate of 2.4 %.Subsequent inactivation of ldh1 gene, encoding the main LDH (Rico et al., 2008) promoted an increment in the conversion rate (4.3 %), suggesting that the engineered route provides an alternative pathway for NAD + regeneration.Once glucose was depleted, reutilization of the produced sorbitol by L. casei recombinant strains was avoided by deleting gutB gene that encodes the IIBC component of the PTS Gut (De Boeck et  al., 2010).L. casei recombinant strains produced mannitol in addition to sorbitol and this polyol mixture was avoided by inactivation of the mtlD gene that encodes a M1PDH.The engineered L. casei strain (lac::gutF ldh1 gutB mtlD) produced sorbitol from lactose, the milk sugar, in non-growing cells or in growing cells under pH control.Fed-batch fermentations using whey permeate, a waste product from the dairy industry with high concentration of lactose, resulted in a conversion rate of 9.4% of lactose into sorbitol (De Boeck et al., 2010).L. plantarum has also been metabolically engineered to produce sorbitol by constitutive overexpression of either srlD1 or srlD2 genes that encode S6PDH activities in a mutant strain deficient in LDH activity (Ladero et al., 2007).Using non-growing or growing cells under pH control resulted in a very efficiency conversion rate of about 65% and 25%, respectively, of sugar into sorbitol.The different efficiencies were suggested to be the result of a higher ATP demand for biomass production in growing cells.

Exopolysaccharides (EPS) production
Some LAB produced EPS, which are extracellular polysaccharides, with important characteristics for the dairy industry.They are used to improve the rheological and textural properties of fermented foods.EPS have also potential as food additives and functional food ingredients.In this sense they are claimed to act as prebiotics in the intestine (Bello et al., 2001) and to stimulate the immune system (Vinderola et al., 2006).The synthesis of EPS in LAB starts at the glycolytic intermediate glucose-6P, which connects the anabolic pathways of biosynthesis of sugar nucleotides, the precursors of the EPS, and the catabolic pathways for obtaining energy through the glycolysis (Figure 1).Glucose-6P is converted to glucose-1P by the -phospoglucomutase (-Pgm) activity, and this sugar phosphate is further metabolized to UDP-glucose and UDP-galactose by the consecutively action of enzymes UDP-glucose pyrophosphorylase (GalU) and UDP-galactose 4-epimerase (GalE) (Boels et al., 2001).Glucose-1P is also substrate for dTDP-glucose pyrophosphorylase to produce dTDPglucose, which will be further metabolized to dTDP-rhanmnose.Glucose, galactose and rhamnose are the principal sugars found in the EPS produced by LAB.The subsequent steps in the synthesis of EPS is the assembly of the monosaccharide repeating unit by specific glycosyltransferases, the polymerization of the repeating units and the secretion from the cell (Welman et al., 2006).The enzymes that participate in these stages are encoded by genes that form part of eps gene clusters.Genetic engineering strategies could be applied to one or more of those stages involved in the EPS biosynthesis in order to increase the EPS production or to modify its composition, however, until now only strains of L. lactis and Streptococcus thermophilus have been genetically modified to enhance EPS biosynthesis.In S. thermophilus the modification of the levels of the GalU, PgmA and the Leloir route enzymes resulted in increased levels of EPS (Levander et al., 2002).Homologous overexpression of a complete eps operon in L. lactis resulted in about fourfold increase in EPS production (Boels et al., 2003).In Lactobacillus species there are no examples of metabolic engineering strategies aimed to produce EPS.In this species the synthesis of EPS has been improved by modifying the culture conditions, such as carbon source and pH.As well, chemically induced mutants of Lactobacillus species that produce higher amounts of EPS than the parental strain have been isolated.The synthesis of EPS by L. casei strain CRL 87 was improved by using galactose as carbon source at a controlled pH of 5.0, and the high EPS production was correlated with high activity level of the enzymes involve in the synthesis of UDP-sugars (Mozzi et al., 2003).Similar approaches were applied for L. helveticus strain ATCC 15807, which produces a higher amount of EPS from lactose at pH 4.5 than at pH 6.2, which was correlated with higher levels of -Pgm activity (Torino et al., 2005).A L. delbrueckii subesp.bulgaricus mutant with improved EPS production has been isolated, and it showed higher amounts of GalU activity, glucose-6P and ATP than the parent strain.These characteristics suggest that GalU and -Pgm enzymes play important roles in the synthesis of high EPS production.The elevated concentration of ATP in the mutant indicated that the glycolysis influence the anabolic route of EPS biosynthesis (Welman et al., 2006).A metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDPgalactose biosynthesis was successfully applied in L. casei.The galU gene coding for GalU enzyme in L. casei strain BL23 was cloned under control of the inducible nisA promoter, and the resulting strain showed about an 80-fold increase in GalU activity, a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose (Rodriguez-Diaz & Yebra, 2011).L. casei s t r a i n B L 2 3 d o e s n o t p r o d u c e E P S , h e n c e i t w o u l d b e a n a d e q ua t e h o s t f o r th e production of heterologous EPS.

Adhesion to intestinal epithelial cells
Adhesion of probiotic bacteria has been employed as a criterion for strain selection and, although it is not indispensable for some probiotic traits, it has positive effects on strain persistence at the gastrointestinal tract and in pathogen inhibition by displacement and competition for adhesion sites.Also, it has been suggested that the capacity to adhere to mucosal surfaces influences the cross-talk established between probiotic bacteria and host cells (Sanchez et al., 2008;Velez et al., 2007).Probiotic strains have shown the ability to bind to intestinal epithelial cultured cells (e.g.Caco-2, HT-29), to mucus components and to proteins of the extracellular matrix (ECM) such as collagen, fibronectin or laminin.Although theses late molecules are not commonly found at the mucosal surface, they may be shed into the mucus or may be exposed in case of trauma or inflammation.They are common targets for pathogen adhesion during the process of infection and adhesion to them by probiotic bacteria can compete with pathogen binding.In contrast to the knowledge about adhesive factors in bacteria causing infectious diseases in humans and animals, the knowledge about adhesion mechanisms in probiotics is very limited.Some molecules from probiotics have been identified as responsible for adhesion, including lipoteichoic acid and exopolysaccharides.However, surface proteins are the major responsible for adherence.Typical surface adhesins from pathogenic bacteria with binding capacity to cultured cells and ECM components are not found in probiotics although it is hypothesized that they may share similar mechanisms for attachment.Similar to some pathogens, probiotic lactobacilli display on their surface moonlighting proteins which are in most cases of cytoplasmic location and are exported and retained on bacterial surfaces by yet unknown mechanisms.These include glycolytic enzymes such as enolase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), molecular chaperones (DnaK, GroEL) and translational elongation factors (EF-Tu, EF-G) (Sanchez et al., 2008;Velez et al., 2007).These proteins have demonstrated binding ability to ECM proteins and epithelial cells and in some cases interfere with host pathways (plasminogen activation mediated by enolase from Lactobacillus crispatus and Lactobacillus johnsonii (Antikainen et al., 2007) or immunoregulation by GroEL from L. johnsonii (Bergonzelli et al., 2006).Other surface proteins with identified roles in adhesion are surface layer (S-layer) proteins or Slp.S-layers from lactobacilli such as L. acidophilus or L. brevis are formed by small basic proteins which form a crystalline matrix on the bacterial surface.The real function of the S-layer is uncertain www.intechopen.combut is has been demonstrated that some Slp proteins display binding capacity to ECM proteins and possess immunoregulatory capabilities.In general the adhesive properties of the above extracellular proteins did not show a strict specificity for substrate binding and it is postulated that they may possess lectin-like characteristics that allow their binding to highly glycosylated ECM proteins and mucosal surfaces (Velez et al., 2007).

Specific proteins implicated in mucus binding
Species of lactobacilli from intestinal origin (L.plantarum, L. reuteri, Lactobacillus gasseri, L. acidophilus, L. johnsonii) express surface proteins covalently anchored to the cell wall by a sortase-dependent mechanism with mucus binding ability (Msa and Mub proteins) (Boekhorst et al., 2006).They are large multidomain proteins containing up to fifteen tandem copies of a mucin-binding domain (MucBP) and act as mannose-dependent adhesins with the capacity to aggregate Saccharomyces cerevisiae cells.The presence of these proteins in certain L. reuteri isolates correlates with the binding ability to mucus.Similarly, the presence in L. plantarum of msa genes is the sole requisite for mucus binding in this species, but domain composition and subtle amino acids changes in each specific Msa protein account for the diverse adhesion properties reported in different strains.In L. rhamnosus GG (LGG), a well characterized probiotic with established mucus adhesion properties, the product of the LGG_02337 gene is the only protein encoded in the genome which contains four MucBP domains about 50 amino acids shorter in length compared to the large mucus binding proteins Msa and Mub.This protein is anchored at the bacterial surface and possesses in vitro binding activity to mucus (von Ossowski et al., 2011).A genome analysis of several L. rhamnosus strains identified the spaCBA cluster as another trait responsible for mucus adherence in LGG.This cluster codes for the three components of pili structures similar to pili described in Gram-positive pathogens that can be identified by immune electron microscopy at the LGG surface (Kankainen et al., 2009).SpaA is the major pilin protein that forms the pilus shaft, while SpaC and SpaB are ancillary pilus proteins which are present at the pilus tip or along the pilus structure and possess adhesive properties.Adhesion experiments with purified proteins, specific antibodies, and mutant construction have demonstrated that SpaC and SpaB are responsible for the mucus binding activity displayed by LGG.This is the first example of the presence of pili adhesive structures in a probiotic strain and exemplifies the adaptation of these bacteria to persist in host tissues.

Engineered lactic acid bacteria with enhanced adhesion
Some of the adhesion factors characterized in probiotic bacteria may be targets for strain engineering aimed to enhance bacterial adhesion.In addition, heterologous expression of well characterized adhesins from different sources can be envisaged.This can be useful to increase residence times at the gastrointestinal tract, enhance interactions with the mucosal immune system and promote competitive exclusion of pathogens by probiotics.Some probiotic strains like L. casei Shirota have been engineered to express a fibronectin binding domain from the Sfb protein of Streptococcus pyogenes, allowing this strain, which barely binds fibronectin, to bind this ECM substrate, fibrinogen and human fibroblasts (Kushiro et al., 2001).However, to date most genetic engineering strategies aimed to increase lactic acid bacteria adhesion have been carried out in the model L. lactis species.This bacterium is not a normal inhabitant of the gastrointestinal tract but it has been used as a food grade delivery vehicle for presenting bioactive molecules to mucosal surfaces, including antigens, cytokines or enzymes.Expression of a protein containing a chitin-binding domain from L. plantarum on the surface of L. lactis resulted in enhanced capacity to attach to natural compounds carrying polymers of N-acetylglucosamine, such as human mucins (Sanchez et al., 2011).The recombinant strain also showed increased attachment to epithelial Caco-2 cells.In another approach, the receptor binding domain of FedD adhesin from E. coli F18 fimbriae was expressed and anchored to the bacterial surface by creating a fusion with the surface anchoring domain of the L. lactis autolysin AcmA.This fusion protein promoted the binding of L. lactis to porcine intestinal epithelial cells (Lindholm et al., 2004).Finally, expression in L. lactis of either a fibronectin binding protein from Staphylococcus aureus o r I n t e r n a l i n A f r o m Listeria monocytogenes promoted its binding to human epithelial cells and bacterial internalization, providing a tool for DNA delivery into eukaryotic cells (Innocentin et al., 2009).

Inmunomodulation of colonic mucosa
In the case of functional properties of lactobacilli, due to legal restrictions and public opinion attitudes against the use of genetically modified microorganisms, the most general strategy has been the selection of naturally competent probiotic strains, nevertheless, some examples of mutants and genetically engineered strains can be found with specific and improved properties (see below).The molecular mechanisms underlying this process are still unknown, however, intervention studies using probiotics in controlled placebo double blind clinical assays are very abundant and different meta-analysis confirmed that several specific beneficial effects of probiotics pass very stringent examination criteria (Williams, 2010), however, they are costly in time and resources and cannot be used to test a great number of strains.Hence, this review will initially describe the general features that characterise the recognition of bacteria by the mucosa and, then, it will focus on the characterisation of the mechanisms of action and the understanding of the effect of probiotics on model systems, as a mean efficiently select funtional strains.As described in the introduction, the mucosal surface is continuously exposed to both potential pathogens and beneficial commensal microorganisms.This creates a requirement for a homeostatic balance between tolerance and immunity that represents a unique regulatory challenge to the mucosal immune system.Dendritic cells (DCs) in the lamina propria efficiently recognise microbial components from the intestinal lumen through PRRs, TLRs and NLRs.Then, DC migrate to draining lymph nodes, where they have the unique ability to activate and influence functional differentiation of naïve T cells.Signals from DC can determine whether tolerance or active immune responses occur to a particular antigen and furthermore influence whether a T helper (T h ) cells of the type T h 1 (innate immune response), T h 2 (adaptive immune response and allergy), T h 17 or T reg (lymphocyte differentiation) predominates.The DC subtype, whether CD11c+ (myeloid) or CD11c-(plasmacytoid), maturation status, and cytokine production contribute to the type of T cell response.For example, when DCs upregulate the coestimulatory molecules CD80 and CD86, produce IL-12 which contributes a Th1 response, but if DCs produce IL-10 and IL-4, they will promote the generation of a Th2 or regulatory T cells (Hart et al., 2004).Furthermore, intestinal epithelial cells (IECs) are not just a simple physical barrier.They express TLRs as well as intracellular NLRs and they can secrete cytokines and regulatory molecules (TSLP, TGF , IL-10, etc) that regulate cytokine secretion by DCs and macrophages.Therefore, EICs actively participate in the discrimination of both pathogenic and commensal bacteria, they are crucial in triggering lymphocyte differentiation, maintaining intestinal immune homeostasis and mechanisms of innate defense (Artis, 2008).As consequence, commensal bacteria and pathogens are detected at different levels, in IECs, DC and macrophages.Different receptors recognise different bacterial ligands, so that the mucosa would integrate the information to recognise the microorganisms approaching the mucosa.PRRs in IECs and DC binding to bacterial molecular patterns (PAMPs) are expressed at the cell surface (TLR2, TLR4, CD14, TLR5) or in specialised endosomes (TLR3, TLR7, TLR8, TLR9).They can recognise single bacterial ligands or act synergistically to recognise others.As a quick overview, TLR3 recognises double stranded viral RNA, TLR9 hypomethylated CpG bacterial DNA, TLR7 and TLR8 were reported to recognize small imidazoquinoline compounds and TLR4, with the aid of CD14, recognises lipopolysaccharides (LPS) and lipoarabinomannans.Peptidoglycan (PGN) of different grampositive bacteria have been shown to interact with TLR2 (Iwaki et al., 2002 ), however, TLR2 recognises other PAMPs (PGN, lipoteicoic acid, mycobacterial cell walls, protozoan parasite GPI anchors, lipoproteins, glycoproteins, glycolipids, LPS, etc).Furthermore, the complex TLR2/TLR6 recognises dipalmitoylated mycoplasma lipoprotein (MALP2), phenol soluble modulin from Staphylococcus epidermidis and fungal zymosan, and also, TLR2 associated to TLR1 recognises triacylated lipoproteins such as Borrelia burgdorferi OspA (for review see (Qureshi & Medzhitov, 2003)).In addition, cell wall components in lactobacilli and firmicutes are recognized by intracellular pattern-recognition molecules members of the nucleotide-binding oligomerization domain (NOD) family.The NLR family (also called Nod-leucine-rich repeats (LRRs)) are responsible for the signalling response induced by bacterial PGN and bacterial surface components, for instance, Nod1/CARD4 receptor in macrophages recognises Meso-diaminopimelic acid (meso-DAP), Nod2/CARD15 recognises muramyl dipeptide (MDP), Nod2 acting as a general sensor for bacteria, Ipaf/CLAN/CARD12 recognises intracellular flagellin (independently of TLR5, which senses extracellular flagellin) and cryopyrin/PYPAF1/NALP3 recognises bacterial RNA (and endogenous danger signals) and among others (for review, see (Franchi et al., 2006).In particular, Nod2 recognizes a PGN motif present on both Gram-positive and Gram-negative bacteria.

Mechanisms of immunomodulation and probiotic factors involved
Nuclear factor κB (NF-κB) is a transcriptional regulator, or rather a regulator family, that controls the expression of hundreds of genes related to different cellular processes, including innate and adaptive immune responses.NF-κB signalling is the major proinflammatory pathway controlling the expression of cytokines, chemokines, enzymes that produce secondary inflammatory mediators and inhibitors of apoptosis.It is activated by numerous proteins, among them pathogen-associated molecular patterns (PAMPs).Various lactobacilli have been described to inhibit NF-κB activity (Kim et al., 2008;Petrof et al., 2004).Although the initial steps of the process are not known, it was determined that L. casei DN-114.001can maintain intestinal homeostasis after an inflammatory stimulus through a process that controls the ubiquitin/proteasome pathway upstream of I-kB resulting in the stabilization of it, therefore blocking NF-κB for nuclear translocation (Tien et al., 2006).Such effects possibly occur through targeting of multiple effectors and, in some cases, through complementary pathways such as NF-κB and p38 MAPK signaling pathways as shown in L. casei and L. reuteri where they could play important roles in the augmentation of innate immunity (Iyer et al., 2008;Kim et al., 2006).Two proteins of L. rhamnosus GG (p40 and p75) (Yan et al., 2007) have been suggested to preserve the tight junction (TJ) integrity, an effect mediated by the PKC and MAPK pathways.They display antiapoptotic activity, prevent epithelial barrier damage caused by several agents and show in vivo effect by decreasing the susceptibility to dextran sulphate sodium (DSS)-induced colon epithelial injury (Yan et al., 2007).Interestingly, recent studies (Bäuerl et al., 2010) showed that these proteins are active cell wall hydrolases present in the seven genome sequences available for strains in the L. casei-paracasei/rhamnosus group.PGNs are cell wall components that interact with the intracellular receptor Nod2.Their in vivo effect changes with their composition composition, which also varies between strains.In the case of Lactobacillus salivarius Ls33 the muropeptide, M-tri-Lys, acts as ligand that protected mice from colitis in a NOD2dependent but MyD88-independent manner (Macho Fernandez et al., 2011).Two strains of L. reuteri and L. casei, but not L. plantarum, had the ability to prime DC to drive the development of Treg cells which produced increased levels of the antiinflammatory cytokine IL-10 (Smits et al., 2005).This ability was mediated by binding to the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN).Targeting of DC-SIGN by certain probiotic bacteria could explain their beneficial effect in the treatment of several immune diseases, including atopic dermatitis and Crohn's disease.However, a DC-SIGN ligand capacity has been found in an extracellular protein (LP_2145) in L. plantarum WCFS1(patent application WO/2009/035330) and in the surfacelayer protein A (SlpA) from L. acidophilus NCFM, inducing IL-10, IL-4 and decreasing IL12p70 synthesis (proinflammatory) (Konstantinov et al., 2008).There are also experiments that reported the in vivo effect on gene transcription in human volunteers where biopsies were analysed by microarrays.Strains of L. rhamosus GG (Di Caro et al., 2005) and L. plantarum WCFS1 (van Baarlen et al., 2011) were used administrated and a differential expression of analysed, noticing that expression affected mainly to genes involved in the immune and inflammatory response, as well as to genes related to apoptosis, growth and cell differentiation, signalling, adhesion, etc. Remarkably, striking differences were found in the modulation of NF-κB related pathways.

Engineered lactic acid bacteria
An efficient expression of IL-10 was achieved in L. lactis , that showed a striking effect in the remediation of DSS (dextran sodium sulphate) induced colitis in IL-10 -/-mice (Steidler et al., 2000).Then, the genetic manipulation strategy was improved and the human IL-10 encoding gene used to replace the thymidylate synthase gene (thyA) in the bacterial chromosome, which achieved a stable genetic construction and a self contained recombinant strain requiring thymidine to proliferate.This strain was used in a double blind assay on Crohn's disease patients (phase I), proving that the intake of this strain significantly decreased the disease activity (Braat et al., 2006).Other anti-inflammatory and epithelium repairing peptide, such as the trefoil factor, was similarly successful in mice (Vandenbroucke et al., 2004).Mutants obtained by site directed mutagenesis, holding genetic changes precisely introduced, have been tested for improved health features.Teichoic acids (TA) activate NF-B through TLR-2 binding and they are one of the main immunostimulatory components of pathogenic grampositive bacteria.This effect was also observed in L. plantarum NCIMB8826, however, a mutant (dlt) deficient in D-alanylation of TA was much more anti-inflammatory than the parental strain on peripheral blood mononuclear cells and mice (Grangette et al., 2005).
In other cases mutants have been very useful to demonstrate the functional effect of certain cell components.In L. casei Shirota (LcS), a mutant strain was very useful to prove that inhibition of the pro-inflammatory cytokine IL-6, through Nod2/ NF-B, was due to a cell wall-derived polysaccharide-PGN complex (PSPG) (Matsumoto et al., 2009).

Engineered probiotics to delivery bioactive proteins
The ability of lactobacilli to survive on the mucosal surfaces of humans and animals has been utilized for the delivery and presentation of bioactive molecules at these surfaces.These bacteria have several advantages, including their recognised GRAS/QPS status, their capacity to interact with the host at several levels and their public acceptance.Studies of lactobacilli as delivery vehicles have mainly focused on the development of mucosal vaccines.In addition, interleukins have been also co-expressed with antigens in lactobacilli to enhance the immune response.Other applications of Lactobacillus species as delivery systems include anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases.The ability of lactobacilli and other LAB to express these antigens/bioactive molecules at mucosal surfaces have been widely reviewed (Monedero & Pérez-Martínez, 2008;Wells & Mercenier, 2008).Some recent examples include the use of Lactobacillus jensenii strains isolated from human vaginal mucosa for the delivery vehicle of a surface-anchored two-domain CD4 (2D CD4) molecule for the mitigation of heterosexual transmission of HIV (Liu et al., 2008).L. casei was the host to express the viral proteins from porcine rotavirus and porcine parvovirus fused the heat-labile toxin B subunit from Escherichia coli.The results showed that mice responded producing increased levels of anti-viral antibodies (Liu et al., 2011).L. casei Zhang was engineered to stably express the p23 immunodominant surface protein of Cryptosporidium parvum sporozoites.Recombinant L. casei Zhang-p23 was able to activate the mucosal immune system and to elicit specific serum immunoglobulin G (IgG) and mucosal IgA in mice.The expression of cytokines such as IL4, IL6, and IFN-gamma was detected in splenocytes of mice by real-time PCR after oral immunization with this strain (Geriletu et al., 2011).A recombinant L. casei strain secreting biologically active murine interleukin-1has been constructed.This strain was able to induce IL8 secretion in Caco2 cells and IL6 in vivo using a ligated-intestinal-loop assay in mice after oral administration.The increased adjuvant properties of this strain were confirmed after intragastric immunization with heatkilled Salmonella enterica serovar Enteritidis (Kajikawa et al., 2010).Another application of lactobacilli has been their use to express molecules to deliver passive immunity against pathogens, such as single-chain antibodies (lactobodies).Martin and coworkers have been able to construct a series of expression cassettes stably integrated in the chromosome to mediate the secretion or surface display of antibody fragments in Lactobacillus paracasei.These new constructed strains, producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus, showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection (Martin et al., 2011).Lactobacilli can also be engineered to produce an increased immune response against cancer cells.L. rhamnosus GG (LGG) has been used to successfully induce tumor regression in an orthotopic model of bladder cancer.The potential of LGG to induce a directed anti-tumor response was evaluated with modified LGG secreting the prostate specific antigen (PSA) or IL15 and PSA (IL-15-PSA).Recombinant LGG activated neutrophils, induced dendritic cells maturation, T cell proliferation and PSA specific cytotoxic T lymphocytes activity leading to the tumor regression (Kandasamy et al., 2011).
In spite of the dozens of reports describing the capacity of LAB to deliver bioactive molecules to the host mucosa, important issues, such as potential side-effects should be always addressed when working with recombinant LAB.The expression of the Salmonella OmpC in a recombinant L. casei was fortuitously found to reduce the secretion of tumor necrosis factor alpha (TNF-) from murine macrophages (Kajikawa & Igimi, 2009).Another non desired effect of a recombinant Lactobacillus was found when L. acidophilus was engineered to express the interferon- to study the local delivery of this molecule in a colitis mouse model.Surprisingly the administration of the recombinant bacteria secreting IFN- has an immunological effect that resulted in the exacerbation of colitis in this model (McFarland et al., 2011).