**5.1. Production of L-Glutamate by** *Corynebacterium glutamicum*

*C. glutamicum* is a facultatively anaerobic, nonpathogenic, non-motile, biotin-auxotrophic, Gram-positive soil bacterium that was isolated more than 50 years ago in a screen for bacteria that excrete glutamate. Since then, derivative strains of this bacterium and related species have been isolated as glutamate producers, and industrial processes for biological glutamate production have been developed. Annual L-glutamate production is estimated to be over 2 million tons, with a continuously increasing demand (3% per year), above all in developing countries. L-glutamate is mainly used in food as a flavour enhancer.

In addition to its importance in industrial biotechnology, *C. glutamicum* has gained interest as a model organism for the *Corynebacterineae*, an industrially relevant suborder of the actinomycetes. Because of the academic and industrial interest, its physiology and metabolism have been deeply characterised, three research groups have independently determined the genomic sequence of *C. glutamicum* strains [103-105], and global analysis techniques such as proteomics, transcriptomics, metabolomics and metabolic flux analysis have been used to obtain a holistic view of the glutamate production process.

*C. glutamicum* exponentially growing cells do not accumulate glutamate. However, glutamate production and excretion can be easily induced by an astonishing variety of treatments, which allow accumulation of glutamate in the culture medium to a concentration as high as 80 g L-1. These include biotin limitation [106], which was the first identified condition for glutamate overproduction, addition of fatty acid ester surfactants such as Tween 40 (polyoxyethylene sorbitan monopalmitate) or Tween 60 (polyoxyethylene sorbitan monoestearate) [107], addition of certain β-lactam antibiotics such as penicillin [108], use of glycerol-auxotrophic or fatty acid-auxotrophic strains [109], use of temperature-sensitive strains cultured at higher temperature [110], or addition of ethambutol, an inhibitor of cell wall arabinogalactan synthesis [111]. In spite of the apparent diversity of treatments to induce glutamate production and secretion, all of them have features in common and there is a unified view that glutamate production is triggered by environmental conditions that produce damage of the cell surface structures [112-114].

Since *C. glutamicum* is a biotin auxotroph, supplementation of a defined medium with a limiting concentration of biotin in batch cultures results in reduced total biomass and concomitant glutamate production. Biotin is a co-factor of acetyl-CoA carboxylase, the first enzyme for fatty acid biosynthesis, and a reduction of this enzyme activity leads to changes in the fatty acid composition of the membrane. In support of this view, a mutation in *dtsR1*, which codes for the beta subunit of acetyl/propionyl-CoA carboxylase, requires fatty acids for growth and, when limited, overproduces glutamate even when grown with an excess of biotin [109]. On the contrary, amplification of its gene dosage resulted in reduction of glutamate production induced by biotin limitation, detergents or penicillin [115]. Glycerol limitation in mutants unable to produce it provokes similar membrane alterations due to the limitation of membrane lipid precursors. Besides the ubiquitous cell membrane, members of the genus *Corynebacterium*, together with *Mycobacteria* and *Nocardia*, have a special cell envelope structure, which consists of a second lipid layer containing mycolic acids, which has a highly ordered structure and plays an important role in determining solute fluxes [116]. Interestingly, mycolic acids of the outer lipid layer are covalently linked to an arabinogalactan layer, which in turn is covalently linked to the underlying peptidoglycan of the cell wall. This structure may thus be envisioned as one large macromolecule, the mycolyl-arabinogalactan-peptidoglycan complex. Treatments with ethambutol or penicillin clearly affect the structure of the cell envelope. The triggering effect of Tween 40 or Tween 60 (but not Tween 20 or Tween 80) may be due to alterations of the mycolic layer structure [112] although it may also affect the activity of acetyl-CoA carboxylase [113], thus leading to cell envelope alterations.

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 305

ammonium assimilation is very limited, according to the *in vivo* flux analyses [119]. *C. glutamicum* and related glutamate-producing species have GDH, GS and GOGAT. Thus, *C. glutamicum* assimilates ammonium, the preferred carbon source of most bacteria, either via GS/GOGAT or via GDH. As in many other bacteria, GS/GOGAT is the main ammonium assimilation pathway when its concentration is limiting, whilst GDH assimilate most of the ammonium when it is present in concentrations above 5 mM [120]. Under glutamate production conditions, which implies high ammonium concentration, the *gltB* and *gltD* genes encoding the GOGAT subunits, are fully repressed by ammonium and no GOGAT activity is detected [119, 121]. Therefore, the main glutamate producing enzyme in these

Production and excretion of large quantities of glutamate require glutamate export to the culture medium but also modification of metabolic fluxes to produce high amounts of glutamate. Metabolic flux analysis under exponential growth vs. glutamate producing conditions [119, 122-125], indicated significant changes in catabolic pathways (Figure 4). Under non-growing, glutamate-producing conditions, glucose catabolism either via glycolysis or the pentose phosphate pathway was clearly reduced. Additionally, there was a significant redistribution of the fluxes at the 2-OG branch point between the TCA cycle and the glutamate biosynthetic pathway, that reduced the flux towards succinyl CoA formation about 2/3, with a concomitant increase in the reductive aminating reaction catalysed by GDH. The increase of the metabolic flux towards glutamate is not due to an increase in the 2-OG-producing isocitrate dehydrogenase (ICDH) or GDH activities because they remain constant [125]. In fact, increasing either ICDH or GDH activity by overexpressing their

The factor with greatest impact on glutamate production is a reduction of the 2-oxoglutarate dehydrogenase complex (ODHC) activity [123, 125]. During exponential growth, although sufficient GDH specific activity was observed, the flux catalyzed by GDH was very small because the *K*m value of GDH for 2-OG is much higher (approximately 50-fold higher) than that of ODHC. Once the ODHC specific activity was decreased after the triggering signals, 2-OG accumulated, and consequently, glutamate was overproduced by GDH. In spite of the initial report claiming that an *odhA* deletion mutant, which has no ODCH activity, overproduced and excreted glutamate [126], there are a number of reports on *odhA* mutants with contradictory results. It appears that those OdhA mutants that overproduced glutamate had an additional mutation in Ncgl1221 (*yggB*) [113], which clearly indicates that reduction or elimination of ODHC activity is necessary but not sufficient for glutamate

Obviously, GDH activity requires the presence of sufficient concentration of its substrates. Paradoxically, most of the NADPH required for anabolic reactions is generated at the pentose phosphate pathway, which is reduced under these conditions. However, it appears that the reaction catalysed by ICDH provides sufficient NADPH to fulfill the demands of the GDH reaction. Similarly, glutamate production and excretion requires a continuous supply of carbon. This is achieved by an increase in the anaplerotic reactions that produce

coding genes, did not affect glutamate production [123].

conditions is GDH.

overproduction.

It was thought for some time that all these treatments affecting cell envelope structure increased the membrane permeability, which would in turn allow leakage of glutamate. However, it is obvious that a specific carrier-mediated export is required, as the increase in the membrane permeability is specific for glutamate, not for other solutes, and glutamate is still exported against a concentration gradient. The glutamate transporter was identified by characterising mutants that produced and accumulated glutamate. All these mutants had substitution mutations or even an insertion mutation in the NCgl1221 gene, which codes for a product showing homology to mechanosensitive channels such as the *E. coli yggB* gene product [113]. The mutant alleles appear to code for a constitutively opened glutamate channel. Exchange of the wild-type allele for these mutant alleles led to glutamate overproduction and excretion without any inducing treatment, and rendered cells resistant to the L-glutamate analog 4-fluoroglutamate. Overexpressing wild type NCgl1221 did not result in constitutive glutamate excretion but led to increased glutamate production and excretion after the induction treatments. On the contrary, a deletion mutant lacking NCgl1221 could not excrete glutamate [113]. Therefore, opening the glutamate channel appears to be essential for glutamate production and export. In addition, this is the first response of *C. glutamicum* to membrane tension alterations, which triggers all other metabolic adaptations leading to efficient glutamate production. The NCgl1221 (*yggB*) product has 4 transmembrane segments, is located in the cytoplasmic membrane [117] and has been recently shown to work as a mechanosensitive channel able to increase the cell survival rate of *Bacillus subtilis* after osmotic down-shock [118].

Although *C. glutamicum* has other potential ammonium assimilating enzymes such as alanine dehydrogenase or diaminopimelate dehydrogenase, their contribution to ammonium assimilation is very limited, according to the *in vivo* flux analyses [119]. *C. glutamicum* and related glutamate-producing species have GDH, GS and GOGAT. Thus, *C. glutamicum* assimilates ammonium, the preferred carbon source of most bacteria, either via GS/GOGAT or via GDH. As in many other bacteria, GS/GOGAT is the main ammonium assimilation pathway when its concentration is limiting, whilst GDH assimilate most of the ammonium when it is present in concentrations above 5 mM [120]. Under glutamate production conditions, which implies high ammonium concentration, the *gltB* and *gltD* genes encoding the GOGAT subunits, are fully repressed by ammonium and no GOGAT activity is detected [119, 121]. Therefore, the main glutamate producing enzyme in these conditions is GDH.

304 Dehydrogenases

cell envelope alterations.

enzyme for fatty acid biosynthesis, and a reduction of this enzyme activity leads to changes in the fatty acid composition of the membrane. In support of this view, a mutation in *dtsR1*, which codes for the beta subunit of acetyl/propionyl-CoA carboxylase, requires fatty acids for growth and, when limited, overproduces glutamate even when grown with an excess of biotin [109]. On the contrary, amplification of its gene dosage resulted in reduction of glutamate production induced by biotin limitation, detergents or penicillin [115]. Glycerol limitation in mutants unable to produce it provokes similar membrane alterations due to the limitation of membrane lipid precursors. Besides the ubiquitous cell membrane, members of the genus *Corynebacterium*, together with *Mycobacteria* and *Nocardia*, have a special cell envelope structure, which consists of a second lipid layer containing mycolic acids, which has a highly ordered structure and plays an important role in determining solute fluxes [116]. Interestingly, mycolic acids of the outer lipid layer are covalently linked to an arabinogalactan layer, which in turn is covalently linked to the underlying peptidoglycan of the cell wall. This structure may thus be envisioned as one large macromolecule, the mycolyl-arabinogalactan-peptidoglycan complex. Treatments with ethambutol or penicillin clearly affect the structure of the cell envelope. The triggering effect of Tween 40 or Tween 60 (but not Tween 20 or Tween 80) may be due to alterations of the mycolic layer structure [112] although it may also affect the activity of acetyl-CoA carboxylase [113], thus leading to

It was thought for some time that all these treatments affecting cell envelope structure increased the membrane permeability, which would in turn allow leakage of glutamate. However, it is obvious that a specific carrier-mediated export is required, as the increase in the membrane permeability is specific for glutamate, not for other solutes, and glutamate is still exported against a concentration gradient. The glutamate transporter was identified by characterising mutants that produced and accumulated glutamate. All these mutants had substitution mutations or even an insertion mutation in the NCgl1221 gene, which codes for a product showing homology to mechanosensitive channels such as the *E. coli yggB* gene product [113]. The mutant alleles appear to code for a constitutively opened glutamate channel. Exchange of the wild-type allele for these mutant alleles led to glutamate overproduction and excretion without any inducing treatment, and rendered cells resistant to the L-glutamate analog 4-fluoroglutamate. Overexpressing wild type NCgl1221 did not result in constitutive glutamate excretion but led to increased glutamate production and excretion after the induction treatments. On the contrary, a deletion mutant lacking NCgl1221 could not excrete glutamate [113]. Therefore, opening the glutamate channel appears to be essential for glutamate production and export. In addition, this is the first response of *C. glutamicum* to membrane tension alterations, which triggers all other metabolic adaptations leading to efficient glutamate production. The NCgl1221 (*yggB*) product has 4 transmembrane segments, is located in the cytoplasmic membrane [117] and has been recently shown to work as a mechanosensitive channel able to increase the cell

Although *C. glutamicum* has other potential ammonium assimilating enzymes such as alanine dehydrogenase or diaminopimelate dehydrogenase, their contribution to

survival rate of *Bacillus subtilis* after osmotic down-shock [118].

Production and excretion of large quantities of glutamate require glutamate export to the culture medium but also modification of metabolic fluxes to produce high amounts of glutamate. Metabolic flux analysis under exponential growth vs. glutamate producing conditions [119, 122-125], indicated significant changes in catabolic pathways (Figure 4). Under non-growing, glutamate-producing conditions, glucose catabolism either via glycolysis or the pentose phosphate pathway was clearly reduced. Additionally, there was a significant redistribution of the fluxes at the 2-OG branch point between the TCA cycle and the glutamate biosynthetic pathway, that reduced the flux towards succinyl CoA formation about 2/3, with a concomitant increase in the reductive aminating reaction catalysed by GDH. The increase of the metabolic flux towards glutamate is not due to an increase in the 2-OG-producing isocitrate dehydrogenase (ICDH) or GDH activities because they remain constant [125]. In fact, increasing either ICDH or GDH activity by overexpressing their coding genes, did not affect glutamate production [123].

The factor with greatest impact on glutamate production is a reduction of the 2-oxoglutarate dehydrogenase complex (ODHC) activity [123, 125]. During exponential growth, although sufficient GDH specific activity was observed, the flux catalyzed by GDH was very small because the *K*m value of GDH for 2-OG is much higher (approximately 50-fold higher) than that of ODHC. Once the ODHC specific activity was decreased after the triggering signals, 2-OG accumulated, and consequently, glutamate was overproduced by GDH. In spite of the initial report claiming that an *odhA* deletion mutant, which has no ODCH activity, overproduced and excreted glutamate [126], there are a number of reports on *odhA* mutants with contradictory results. It appears that those OdhA mutants that overproduced glutamate had an additional mutation in Ncgl1221 (*yggB*) [113], which clearly indicates that reduction or elimination of ODHC activity is necessary but not sufficient for glutamate overproduction.

Obviously, GDH activity requires the presence of sufficient concentration of its substrates. Paradoxically, most of the NADPH required for anabolic reactions is generated at the pentose phosphate pathway, which is reduced under these conditions. However, it appears that the reaction catalysed by ICDH provides sufficient NADPH to fulfill the demands of the GDH reaction. Similarly, glutamate production and excretion requires a continuous supply of carbon. This is achieved by an increase in the anaplerotic reactions that produce oxaloacetate. In *C. glutamicum* two reactions can yield oxaloacetate. One is the standard anaplerotic reaction catalysed by the phosphoenol pyruvate carboxylase (PEPc). The other is pyruvate carboxylase (Pc), which catalyses oxaloacetate production from pyruvate. Metabolic flux of PEPc remains constant in both conditions whilst the Pc flux, undetectable during exponential growth, is clearly increased in the glutamate-producing conditions upon addition of Tween-40 [124]. This suggests a relevant role of Pc in providing sufficient oxaloacetate for glutamate production. Despite this evidence, pyruvate carboxylase cannot be relevant for glutamate production when induced by biotin limitation, as biotin is also a prosthetic group of Pc. As expected, *pyc* disruptants lacking Pc can produce and excrete glutamate as the wild type strain [127], indicating that PEPc may be sufficient to provide the required carbon under this particular inducing regime.

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 307

How is ODHC activity decreased during glutamate production? ODHC is a complex composed of three different subunits: OdhA (E1), SucB (E2) and LpdA (E3). Many TCA cycle enzymes are regulated at transcriptional or posttranscriptional levels [128]. Activity of this complex is controlled by OdhI, a small protein (15,4 KDa) that binds OdhA (the E1 subunit). Induced glutamate excretion is virtually abolished in a OdhI deletion mutant, thus indicating that inhibition of ODHC activity through OdhI is critical for glutamate overproduction [129]. The OdhI function is regulated by phosphorylation by PknG, which phosphorylates and inactivates OdhI [130], and dephosphorylation by Ppp. PknG deletion mutants showed higher glutamate production when induced by some treatments [129],

The main question is why do *C. glutamicum* cells excrete large amounts of L-glutamate when exposed to the mentioned induction treatments? Certainly, it does not appear that it is a response to metabolic changes since the inducing treatments do not imply changes in nutritional status. Since these treatments all affect cell surface structures and may therefore alter membrane tension, L-glutamate production may be the response to this membrane stress, as glutamate might function as a compatible solute to prevent cells from bursting. Thus, L-glutamate production by *C. glutamicum* may be a mechanism of adaptation to environmental changes affecting cell surface structures that starts by opening a glutamate export mechanosensitive channel, which in turn somehow triggers the metabolic adaptations (reduction of pentose phosphate pathway, reduction of ODHC activity, increase of the anaplerotic reactions) required to produce and excrete high amounts of glutamate. A similar role of L-glutamate in osmoprotection was also described for *E. coli* [131], and confirmed by the osmosensitive phenotype provoked by mutations unable to activate *gdhA* in a GOGAT deficient background during osmotic upshift [4]. However, there are no reports on metabolic adaptations leading to glutamate production and excretion upon osmotic

which is consistent with its role in controlling ODHC activity.

**5.2. Aroma and flavour production by Lactic Acid Bacteria (LAB)** 

Accelerating or diversifying flavour development in cheese is of major economical interest since final flavour of cheeses partly determines the consumer's choice. Flavour formation occurs during cheese ripening, and is associated to non-starter lactic acid bacteria (NSLAB), adventitious microflora that occur in the milk or appear later during cheese manufacturing. After characterisation, some strains have been selected as flavour-producing adjunts and shown that cheeses with these adjunts are richer in free amino acids and have enhanced flavour intensity [132]. However, flavour development is a time consuming and expensive process that is still not well mastered, and selection of strains for cheese ripening is still an empirical process based on cheese trials with different strains and sensory analyses, that has

Amino acid catabolism, particularly that of aromatic amino acids, branched chain amino acids, and methionine, is a major player in flavour formation in cheese, especially in cheeses containing only LAB. Amino acid conversion to aroma compounds proceeds by two

shock in this bacterium.

had varying success [133].

**Figure 4.** Metabolic fluxes from glucose to glutamate under vegetative growth or non-growing glutamate-producing conditions. Red arrows show metabolic fluxes reduced during glutamate production. Green arrows show those reactions that appear or are increased under the same condition.

How is ODHC activity decreased during glutamate production? ODHC is a complex composed of three different subunits: OdhA (E1), SucB (E2) and LpdA (E3). Many TCA cycle enzymes are regulated at transcriptional or posttranscriptional levels [128]. Activity of this complex is controlled by OdhI, a small protein (15,4 KDa) that binds OdhA (the E1 subunit). Induced glutamate excretion is virtually abolished in a OdhI deletion mutant, thus indicating that inhibition of ODHC activity through OdhI is critical for glutamate overproduction [129]. The OdhI function is regulated by phosphorylation by PknG, which phosphorylates and inactivates OdhI [130], and dephosphorylation by Ppp. PknG deletion mutants showed higher glutamate production when induced by some treatments [129], which is consistent with its role in controlling ODHC activity.

306 Dehydrogenases

oxaloacetate. In *C. glutamicum* two reactions can yield oxaloacetate. One is the standard anaplerotic reaction catalysed by the phosphoenol pyruvate carboxylase (PEPc). The other is pyruvate carboxylase (Pc), which catalyses oxaloacetate production from pyruvate. Metabolic flux of PEPc remains constant in both conditions whilst the Pc flux, undetectable during exponential growth, is clearly increased in the glutamate-producing conditions upon addition of Tween-40 [124]. This suggests a relevant role of Pc in providing sufficient oxaloacetate for glutamate production. Despite this evidence, pyruvate carboxylase cannot be relevant for glutamate production when induced by biotin limitation, as biotin is also a prosthetic group of Pc. As expected, *pyc* disruptants lacking Pc can produce and excrete glutamate as the wild type strain [127], indicating that PEPc may be sufficient to provide the

**Figure 4.** Metabolic fluxes from glucose to glutamate under vegetative growth or non-growing glutamate-producing conditions. Red arrows show metabolic fluxes reduced during glutamate production. Green arrows show those reactions that appear or are increased under the same condition.

required carbon under this particular inducing regime.

The main question is why do *C. glutamicum* cells excrete large amounts of L-glutamate when exposed to the mentioned induction treatments? Certainly, it does not appear that it is a response to metabolic changes since the inducing treatments do not imply changes in nutritional status. Since these treatments all affect cell surface structures and may therefore alter membrane tension, L-glutamate production may be the response to this membrane stress, as glutamate might function as a compatible solute to prevent cells from bursting. Thus, L-glutamate production by *C. glutamicum* may be a mechanism of adaptation to environmental changes affecting cell surface structures that starts by opening a glutamate export mechanosensitive channel, which in turn somehow triggers the metabolic adaptations (reduction of pentose phosphate pathway, reduction of ODHC activity, increase of the anaplerotic reactions) required to produce and excrete high amounts of glutamate. A similar role of L-glutamate in osmoprotection was also described for *E. coli* [131], and confirmed by the osmosensitive phenotype provoked by mutations unable to activate *gdhA* in a GOGAT deficient background during osmotic upshift [4]. However, there are no reports on metabolic adaptations leading to glutamate production and excretion upon osmotic shock in this bacterium.

### **5.2. Aroma and flavour production by Lactic Acid Bacteria (LAB)**

Accelerating or diversifying flavour development in cheese is of major economical interest since final flavour of cheeses partly determines the consumer's choice. Flavour formation occurs during cheese ripening, and is associated to non-starter lactic acid bacteria (NSLAB), adventitious microflora that occur in the milk or appear later during cheese manufacturing. After characterisation, some strains have been selected as flavour-producing adjunts and shown that cheeses with these adjunts are richer in free amino acids and have enhanced flavour intensity [132]. However, flavour development is a time consuming and expensive process that is still not well mastered, and selection of strains for cheese ripening is still an empirical process based on cheese trials with different strains and sensory analyses, that has had varying success [133].

Amino acid catabolism, particularly that of aromatic amino acids, branched chain amino acids, and methionine, is a major player in flavour formation in cheese, especially in cheeses containing only LAB. Amino acid conversion to aroma compounds proceeds by two different pathways: (i) elimination reactions catalysed by amino acid lyases that produce different alcohols, and (ii) transamination reactions leading to different 2-oxoacids. The latter is the major pathway in LAB. The resulting 2-oxoacids are not responsible for flavour production but are then transformed to aldehydes, alcohols, carboxylic acids, hydroxy acids or methanethiol by additional steps that may be catalysed by the 2-oxoacid-producing LAB or by the inoculated starter LABs [134, 135]. Figure 5 shows the transaminations and further reactions leading to aroma compounds. It appears that the different flavours produced by different LAB strains depend on the proportion of the different 2-oxoacid produced and, therefore, on the relative aminotransferases activities [136].

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 309

strain of *L. lactis*, which

of this, GDH can only be used for glutamate catabolism, and this reaction constitutes the major source of 2-OG in these strains. As shown in Fig. 5, the GDH role in aroma development by LAB in cheeses is to catalyse the oxidative deamination of glutamate in order to replenish the 2-OG consumed by the transamination reactions. The importance of GDH in aroma production was initially shown by cloning and heterologous expression of

resulted in an increase of amino acids degradation and, more importantly, an increase in the proportion of carboxylic acids, which are important aroma compounds [141]. This result is the proof of concept that GDH can substitute the exogenous 2-OG. Other reports describing the effect of GDH on aroma production have followed, including the natural transfer by conjugation of the *gdh* gene coded in the pGDH442 plasmid among LAB strains [142]. The relevance of this is that the resulting transconjugants are not considered genetically

An evident correlation between GDH activity and aroma production has been established for both mesophilic and thermophilic LAB [143, 144]. Because of this, the presence of GDH activity has been proposed as a criterion to select flavour-producing LAB strains for cheese ripening. The use of different LAB strains with different aminotransferases especificities together with the use of GDH to enhance the transamination reactions may represent an

*Centro Andaluz de Biología del Desarrollo, CSIC-Universidad Pablo de Olavide-Junta de Andalucía,* 

We are very grateful to Nuria Pérez and Guadalupe Martín-Cabello for their technical help and all members of the laboratory for their insights and suggestions. Work in the authors' laboratories is supported by the Spanish Ministry of Economy and Competitivity, grants BIO2008-01805, BIO2010-17853, BIO2011-24003 and CSD2007-00005, and by the Andalusian

[1] Smith EL, Austen, BM, Blumenthal, KM, Nyc, JF (1975) Glutamate dehydrogenases. In:

*Department of Microbiology, Faculty for Biology. University of Freiburg, Freiburg, Germany* 

modified organisms and can be used in the food industry as starters or adjuncts.

the GDH gene from *Peptostreptococcus asaccharolyticus* into a GDH-

effective way of intensifying and diversifying cheese aromas.

government, grants P05-CVI-131 and P07-CVI-2518.

Boyer PD, editor. The Enzymes: Academic Press. p. 293–367.

, Ines Canosa and Fernando Govantes

**Author details** 

Eduardo Santero\*

*Seville, Spain* 

Ana B. Hervás

**6. References** 

Corresponding Author

 \*

**Acknowledgement** 

**Figure 5.** Main amino acid catabolic pathways in LAB leading to aroma compounds (boxed). Doted arrows represent chemical reactions.

Although lactococci have high aminotransferase activity, only very low and slow amino acid degradation occurs. Cheeses are rich in amino acids and peptides but the concentration of 2- OG is low. Supplementation of several types of cheeses with 2-OG led to a decrease in the levels of amino acids important for aroma development, which indicated that catabolism of these amino acids had been enhanced [137, 138], and sensory analyses indicated that addition of 2-OG resulted in changes in creamy character, aroma intensity and fruity notes. These results clearly established that 2-OG availability is the limiting factor for the transamination reactions that convert aminoacids to aroma compounds and prompted scientist to look for sources of 2-OG.

Ocurrence of GDH in starter and non-starter LAB is heterogeneous, as just a few LAB strains show GDH activity. Some strains of the starter *Lactococcus lactis* have GDH activity but it is coded in a plasmid (pGDH442) rather than in its chromosome [139]. However, these GDH+ strains are glutamate auxotrophs and cannot synthesize glutamate because of 2-OG limitation. The oxidative TCA cycle in these strains is interrupted at the isocitrate dehydrogenase step [140], thus blocking the major source of 2-OG for most bacteria. Because of this, GDH can only be used for glutamate catabolism, and this reaction constitutes the major source of 2-OG in these strains. As shown in Fig. 5, the GDH role in aroma development by LAB in cheeses is to catalyse the oxidative deamination of glutamate in order to replenish the 2-OG consumed by the transamination reactions. The importance of GDH in aroma production was initially shown by cloning and heterologous expression of the GDH gene from *Peptostreptococcus asaccharolyticus* into a GDH strain of *L. lactis*, which resulted in an increase of amino acids degradation and, more importantly, an increase in the proportion of carboxylic acids, which are important aroma compounds [141]. This result is the proof of concept that GDH can substitute the exogenous 2-OG. Other reports describing the effect of GDH on aroma production have followed, including the natural transfer by conjugation of the *gdh* gene coded in the pGDH442 plasmid among LAB strains [142]. The relevance of this is that the resulting transconjugants are not considered genetically modified organisms and can be used in the food industry as starters or adjuncts.

An evident correlation between GDH activity and aroma production has been established for both mesophilic and thermophilic LAB [143, 144]. Because of this, the presence of GDH activity has been proposed as a criterion to select flavour-producing LAB strains for cheese ripening. The use of different LAB strains with different aminotransferases especificities together with the use of GDH to enhance the transamination reactions may represent an effective way of intensifying and diversifying cheese aromas.
