**2. Lactic acid production**

148 Food Industrial Processes – Methods and Equipment

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 In order to developed a potential food-grade expression system the erythromycin gene in the pSIP vectors has been replaced by the *alr* gene, which encodes the alanine racemase enzyme that is essential for cell wall biosynthesis (Nguyen *et al.*, 2011).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;

**1.3.2 DNA mutagenesis systems: integration and insertion systems, and random** 

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.*,

Perez-Arellano & Perez-Martinez, 2003).

**mutagenesis systems** 

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

bacteria and yeasts have demonstrated that biotechnological production may represent an

145

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

Fig. 2. Proposed pathways for sorbitol, mannitol, acetoin and diacetyl production by engineered lactic acid bacteria. [1] phosphoenolpyruvate: lactose phosphotransferase

phosphogalactosidase, [4] glucokinase, [5] phosphoglucose isomerase, [6] sorbitol-6P dehydrogenase, [7] mannitol-1P dehydrogenase, [8] phosphofructokinase, [9] fructose 1,6 bisP aldolase, [10] galactose-6P isomerase, [11] tagatose-6P kinase, [12] tagatose-1,6DP aldolase, [13] triose-phosphate isomerase, [14] lactate dehydrogenase, [15]-acetolactate synthase, [16] acetohydroxyacid synthase , [17]-acetolactate decarboxylase, [18] pyruvate oxidase, [19] pyruvate dehydrogenase complex, [20] pyruvate-formate lyase. DHAP,

system, [2] phosphoenolpyruvate: glucose phosphotransferase system, [3] -

dihydroxiacetone phosphate.

efficient and cost-effective alternative to the chemical production.

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).
