**Genetically Engineered Lactobacilli for Technological and Functional Food Applications**

María J. Yebra, Vicente Monedero, Gaspar Pérez-Martínez and Jesús Rodríguez-Díaz *Departamento de Biotecnología de los Alimentos IATA- CSIC Spain* 

### **1. Introduction**

142 Food Industrial Processes – Methods and Equipment

Zaidi, A., J.L. Gainer, G. Carta, A. Mrani, T. Kadiri, Y. Belarbi and A. Mir, (2002)

parameters and chain-length effects , *J. Biotechnol.* 93, pp. 209–216.

136

Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic

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.

#### **1.1 Metabolic potential of lactobacilli**

Because of their fermentative metabolism and global utilization in food fermentations, LAB are specially suited for rerouting sugar metabolism to produce industrially important food compounds. During fermentation, monosaccharides are catabolized through glycolysis (Embden-Meyerhoff pathway) and related pathways (Figure 1). Glycolytic catabolism of sugars involves phosphorylation reactions that drive hexoses to fructose-1,6-bisP, which is then hydrolysed to glyceraldehyde-3P (GADH-3P) and dihydroxyacetone-P. Then, GADH-

Fig. 1. Scheme of glycolysis, phosphoketolase pathway and anabolic pathway of UDPsugars in LAB. [1] glucokinase, [2] phosphoglucose isomerase, [3] phosphofructokinase, [4]

dehydrogenase, [7] phosphoglycerate kinase, [8] pyruvate kinase, [9] lactate dehydrogenase, [10] hexokinase, [11] glucose-6P dehydrogenase, [12] 6-phosphogluconate dehydrogenase, [13] ribulose-5P 3-epimerase, [14] xylulose-5P phosphoketolase, [15] phosphotransacetylase, [16] acetaldehyde dehydrogenase, [17] alcohol dehydrogenase, [18] acetate kinase, [19] phosphoglucomutase, [20] UDP-glucose pyrophosphorylase, [21] UDP-galactose 4 epimerase, [22] deoxyTDP-glucose pyrophosphorylase, [23] deoxyTDP-glucose 4,6-

139

phosphotransferase system; Per: permease; CoA, coenzyme A; DHAP, dihydroxiacetone phosphate; GADH-3P, glyceraldehyde-3-phosphate; UDP-Glu, UDP-glucose; UDP-Gal, UDP-galactose; dTDP-Glu, deoxyTDP-glucose; dTDP-kdMan, deoxyTDP-4-keto-6-

fructose 1,6-bisP aldolase, [5] triose-phosphate isomerase, [6] glyceraldehyde-3P

dehydratase, [24] deoxyTDP-rhamnose synthetic enzyme system. PTS, PEP

deoxymannose; EPS, exopolysaccharide; PEP, phospho*enol*pyruvate.

3P undertakes dephosphorylation and oxidation, which yields 2 pyruvate, 2 ATP and the reduction of 2 NAD+ to 2 NADH per glucose. Under normal metabolic conditions NADH is used mainly to reduce pyruvate. Some LAB exclusively produce lactate from pyruvate (homofermentative), while other species (heterofermentative) produce lactate, acetate, ethanol and CO2. There are other differences in heterofermentative species, since they can shift sugar catabolism towards the so called pentose phosphate or phosphoketolase pathway that renders GADH-3P and acetyl-P. Then, GADH-3P enters the lower part of the glycolysis. In this pathway two additional NADH molecules are oxidised by means of alcohol dehydrogenase that produces ethanol from acetyl-CoA. Therefore, the global balance of the heterofermentation of one mol of glucose is one mol of lactate, one mol of ethanol and one mol of CO2, with a net energy yield of one mol of ATP.

An important strategy frequently used during metabolic engineering consists in blocking the formation of natural proton sinks, such as lactate or ethanol in the final steps of glycolysis. However, dissipation of the H+ pool has such a great relevance that LAB normally has several isoenzymes of L-lactate dehydrogenase (L-LDH) as showed by the analysis of different genomes such as *Lactobacillus plantarum* and *Lactobacillus casei*  (Kleerebezem *et al.*, 2003; Rico *et al.*, 2008), and alternative dehydrogenases yielding Dlactate have been found in most LAB genomes. An additional difficulty is imposed by the fact that glycolysis is subject to a strict allosteric regulation by its own intermediate and final metabolites, as well as by Pi, fructose-1,6bisP, phospho*enol*pyruvate (PEP), ADP, ATP and NADH/NAD+ ratio. Its robustness and flexibility would assure an efficient bacterial growth, so that its activity rate would always respond to the cell's energy demand. It has also been observed that there are three enzyme activities especially sensitive to allosteric modulation, which are most relevant in the pathway's regulation and they are: phosphofructokinase (PFK), GADH-3P dehydrogenase and pyruvate kinase. PFK is strongly inhibited by PEP and pyruvate kinase activity is inhibited by Pi in *Lactococcus lactis* and this enzyme is stimulated by ADP and fructose-1,6bisP in *Lactobacillus bulgaricus* (Branny *et al.*, 1998). Furthermore, in *L. lactis*, GADH-3P dehydrogenase has a remarkable role in the modulation of the carbon flux, which is regulated by the NADH/NAD+ ratio, as it happens with LDH (Garrigues *et al.*, 1997).

LAB suitability as starter cultures in dairy fermentations highly depends on their ability to produce small concentrations of volatile compounds derived from the alternative metabolism of pyruvate. The production of diacetyl and acetoin is quite common in LAB. These compounds are produced through decarboxylation of -acetolactate obtained from pyruvate by the enzyme -acetolactate syntase (Figure 2). Additionally, under substrate limitation and anaerobiosis, the enzyme pyruvate-formate lyase produces acetyl-CoA and formate from pyruvate and CoA. Acetyl-CoA is an important metabolite, as it can be used as electron acceptor to oxidise NADH or as energy compound to obtain ATP. The enzymatic complex of pyruvate dehydrogenase also produces acetyl-CoA, CO2 and NADH from pyruvate, coenzyme A and NAD+. Under aerobic conditions, this is an anabolic enzyme producing acetyl-CoA used for lipid synthesis, but under aerobic conditions, it also has a catabolic function where NADH oxidases can regenerate the excess of NADH produced. Pyruvate oxidase mediates onversion of pyruvate to acetyl-P, CO2 and H2O2. This activity allows to obtain ATP when carbon sources are limiting, by substrate level phosphorylation of acetyl-P.

3P undertakes dephosphorylation and oxidation, which yields 2 pyruvate, 2 ATP and the reduction of 2 NAD+ to 2 NADH per glucose. Under normal metabolic conditions NADH is used mainly to reduce pyruvate. Some LAB exclusively produce lactate from pyruvate (homofermentative), while other species (heterofermentative) produce lactate, acetate, ethanol and CO2. There are other differences in heterofermentative species, since they can shift sugar catabolism towards the so called pentose phosphate or phosphoketolase pathway that renders GADH-3P and acetyl-P. Then, GADH-3P enters the lower part of the glycolysis. In this pathway two additional NADH molecules are oxidised by means of alcohol dehydrogenase that produces ethanol from acetyl-CoA. Therefore, the global balance of the heterofermentation of one mol of glucose is one mol of lactate, one mol of

An important strategy frequently used during metabolic engineering consists in blocking the formation of natural proton sinks, such as lactate or ethanol in the final steps of glycolysis. However, dissipation of the H+ pool has such a great relevance that LAB normally has several isoenzymes of L-lactate dehydrogenase (L-LDH) as showed by the analysis of different genomes such as *Lactobacillus plantarum* and *Lactobacillus casei*  (Kleerebezem *et al.*, 2003; Rico *et al.*, 2008), and alternative dehydrogenases yielding Dlactate have been found in most LAB genomes. An additional difficulty is imposed by the fact that glycolysis is subject to a strict allosteric regulation by its own intermediate and final metabolites, as well as by Pi, fructose-1,6bisP, phospho*enol*pyruvate (PEP), ADP, ATP and NADH/NAD+ ratio. Its robustness and flexibility would assure an efficient bacterial growth, so that its activity rate would always respond to the cell's energy demand. It has also been observed that there are three enzyme activities especially sensitive to allosteric modulation, which are most relevant in the pathway's regulation and they are: phosphofructokinase (PFK), GADH-3P dehydrogenase and pyruvate kinase. PFK is strongly inhibited by PEP and pyruvate kinase activity is inhibited by Pi in *Lactococcus lactis* and this enzyme is stimulated by ADP and fructose-1,6bisP in *Lactobacillus bulgaricus* (Branny *et al.*, 1998). Furthermore, in *L. lactis*, GADH-3P dehydrogenase has a remarkable role in the modulation of the carbon flux, which is regulated by the NADH/NAD+ ratio, as it happens

LAB suitability as starter cultures in dairy fermentations highly depends on their ability to produce small concentrations of volatile compounds derived from the alternative metabolism of pyruvate. The production of diacetyl and acetoin is quite common in LAB. These compounds are produced through decarboxylation of -acetolactate obtained from pyruvate by the enzyme -acetolactate syntase (Figure 2). Additionally, under substrate limitation and anaerobiosis, the enzyme pyruvate-formate lyase produces acetyl-CoA and formate from pyruvate and CoA. Acetyl-CoA is an important metabolite, as it can be used as electron acceptor to oxidise NADH or as energy compound to obtain ATP. The enzymatic complex of pyruvate dehydrogenase also produces acetyl-CoA, CO2 and NADH from pyruvate, coenzyme A and NAD+. Under aerobic conditions, this is an anabolic enzyme producing acetyl-CoA used for lipid synthesis, but under aerobic conditions, it also has a catabolic function where NADH oxidases can regenerate the excess of NADH produced. Pyruvate oxidase mediates onversion of pyruvate to acetyl-P, CO2 and H2O2. This activity allows to obtain ATP when carbon sources are limiting, by substrate level phosphorylation

ethanol and one mol of CO2, with a net energy yield of one mol of ATP.

with LDH (Garrigues *et al.*, 1997).

of acetyl-P.

Fig. 1. Scheme of glycolysis, phosphoketolase pathway and anabolic pathway of UDPsugars in LAB. [1] glucokinase, [2] phosphoglucose isomerase, [3] phosphofructokinase, [4] fructose 1,6-bisP aldolase, [5] triose-phosphate isomerase, [6] glyceraldehyde-3P dehydrogenase, [7] phosphoglycerate kinase, [8] pyruvate kinase, [9] lactate dehydrogenase, [10] hexokinase, [11] glucose-6P dehydrogenase, [12] 6-phosphogluconate dehydrogenase, [13] ribulose-5P 3-epimerase, [14] xylulose-5P phosphoketolase, [15] phosphotransacetylase, [16] acetaldehyde dehydrogenase, [17] alcohol dehydrogenase, [18] acetate kinase, [19] phosphoglucomutase, [20] UDP-glucose pyrophosphorylase, [21] UDP-galactose 4 epimerase, [22] deoxyTDP-glucose pyrophosphorylase, [23] deoxyTDP-glucose 4,6 dehydratase, [24] deoxyTDP-rhamnose synthetic enzyme system. PTS, PEP phosphotransferase system; Per: permease; CoA, coenzyme A; DHAP, dihydroxiacetone phosphate; GADH-3P, glyceraldehyde-3-phosphate; UDP-Glu, UDP-glucose; UDP-Gal, UDP-galactose; dTDP-Glu, deoxyTDP-glucose; dTDP-kdMan, deoxyTDP-4-keto-6 deoxymannose; EPS, exopolysaccharide; PEP, phospho*enol*pyruvate.

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

141

aggressions, while preserving homeostasis (Sansonetti & Medzhitov, 2009).

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 pAM1 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

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

**1.3 Tools and strategies for genetic manipulation of lactobacilli 1.3.1 Gene cloning vectors, genetic markers and promoters** 

cell wall (Reveneau *et al.*, 2002).

#### **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, H2O2), 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

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, H2O2), 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 heat-

**1.2 Functional properties of lactobacilli** 

shock 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).

#### **1.3 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 pAM1 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

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

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

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 *ldh*s 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

*ldh1 ldh2*,

*ldh1 hicD* double mutant. *ldh2*, *ldh3* or *ldh4*

*ldh1 ldh3*,

*ldh1 ldh4*) had a

143

transposon mutagenesis (Lee *et al.*, 2010).

activity of HicD, since it was abolished in a

single mutations or combined with an *ldh1* deletion (

starch or cellulosic compounds, respectively (Okano *et al.*, 2010).

**2. Lactic acid production** 

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).

#### **1.3.2 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).
