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Applied and Environmental Microbiology, August 2008, p. 4590-4600, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00150-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria{triangledown} ,{dagger}

Mengjin Liu,1,2* Arjen Nauta,2 Christof Francke,1,4 and Roland J. Siezen1,3,4

Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, The Netherlands,1 Friesland Foods Corporate Research, Deventer, The Netherlands,2 NIZO food research, Ede, The Netherlands,3 TI Food and Nutrition, Wageningen, The Netherlands4


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INTRODUCTION
 
Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when LAB are growing in milk. One of the main precursors for flavor compounds in milk is casein, although they can also be derived from fatty acids and sugars. The proteolytic system of LAB degrades casein into its constituent amino acids, which can be converted to flavor compounds. Although amino acid catabolism by LAB has been well researched (5, 31, 65, 78), many flavor-forming routes are yet to be discovered.

Over 20 LAB genomes have been fully sequenced (1, 11, 12, 16, 19, 41, 44, 45, 53, 68, 71). The available genomic information provides us with new opportunities to study the flavor-forming potential of LAB. However, one of the main problems that one encounters while reconstructing flavor-forming routes based on the genomic information stored in the public databases is the inconsistency in the functional annotation for many of the relevant genes. These genes are mostly members of larger protein families. Moreover, even when the functional annotation in databases is appropriate, it sometimes reflects only part of the protein's full functional potential, since broad substrate specificities are often not taken into consideration in the annotation.

We have now improved the functional annotation of the key enzymes in the formation of flavor compounds from amino acids by applying comparative genomics approaches that have been developed within our group to specify the annotation of homologous proteins, by combining phylogeny, gene context, and experimental evidence (32). We focused especially on the enzymes involved in the metabolism of sulfur-containing amino acids since these are known to be precursors of many flavor compounds in dairy fermentations. Comparative analysis of the various sequenced LAB species and strains resulted in an overall view of differences in their flavor-forming capacities.


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IN SILICO ANALYSES OF LAB GENOMES
 
Complete LAB genome sequences were obtained from the NCBI microbial genome database (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) on 30 November 2007: Lactobacillus acidophilus NCFM (identified as LAC in the tables and figures; accession code CP000033), Lactobacillus johnsonii NCC 533 (LJO; AE017198), Lactobacillus gasseri ATCC 33323 (LGA; CP000413), Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (LDB; CR954253), L. delbrueckii subsp. bulgaricus ATCC BAA365 (LBU; CP000412), Lactobacillus plantarum WCFS1 (LPL; AL935263), Lactobacillus brevis ATCC 367 (LBE; CP000416), Lactobacillus sakei 23K (LSK; CR936503), Lactobacillus salivarius UCC118 (LSL; CP000233), Oenococcus oeni PSU1 (OOE; CP000411), Pediococcus pentosaceus ATCC 25745 (PPE, CP000422), Leuconostoc mesenteroides ATCC 8293 (LME; CP000414), Lactobacillus casei ATCC 334 (LCA; CP000423), Lactococcus lactis subsp. lactis IL1403 (LLX; AE005176), Lactococcus lactis subsp. cremoris MG1363 (LLM; AM406671), L. lactis subsp. cremoris SK11 (LLA; CP000425), Streptococcus thermophilus CNRZ1066 (STH; CP000024), S. thermophilus LMG18311 (STU; CP000023), S. thermophilus LMD9 (STM; CP000419), and Lactobacillus reuteri F275 (LRF; CP000705). The complete genome sequence of L. reuteri JCM1112 (LRE) was obtained from the ERGO database (50). The phylogeny of these LAB is shown in Fig. 1.


Figure 1
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  		FIG. 1. Phylogenetic tree of LAB constructed on the basis of concatenated alignments of four subunits ({alpha}, β, β', and {delta}) of the DNA-dependent RNA polymerase. Different colors represent the various origins or usages of LAB: red for dairy fermentation, blue for other fermentation such as beer, wine, plants, or meat, and green for gastrointestinal tract bacteria. The L. acidophilus group is boxed.

Protein sequences of experimentally verified enzymes involved in flavor formation were obtained from the Uniprot database (9). These sequences were used to perform BLAST searches against all LAB genomes (3). In addition, hidden Markov models (HMMs) of the related protein families were obtained from the Pfam database (10) and used to identify homologs with the HMMER 2.3.2 package (24). Homologous sequences from each enzyme family derived from both BLAST and HMM searches were collected, and the redundant sequences were removed. Orthologous relationships between the various homologs were determined on the basis of phylogeny and synteny. For each protein family, a multiple sequence alignment was created with MUSCLE (25). The alignments included representative sequences from literature for which experimental evidence is available. CLUSTAL W was used to generate bootstrapped (n = 1,000) neighbor-joining trees on the basis of the multiple sequence alignments (67). The trees were visualized in LOFT (69), a tool that automatically identifies orthologous groups. When possible, gene context was used to distinguish between functional variants. The gene context was studied by using the ERGO Bioinformatics Suite (50). The complete procedure is depicted in Fig. S1 in the supplemental material.


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FLAVOR-FORMING PATHWAYS FROM AMINO ACIDS
 
Free amino acids produced by proteolysis are converted to various flavor compounds through amino acid catabolism. The branched-chain amino acids (valine, leucine, and isoleucine), the aromatic amino acids (tyrosine, tryptophan, and phenylalanine), and the sulfur-containing amino acids (methionine and cysteine) are the main amino acid sources for flavor compounds. The conversion of these amino acids into flavors proceeds via two distinct routes: transamination and elimination (Fig. 2 and Fig. 3) (5, 65, 78). The transamination route is initiated by aminotransferases that convert amino acids into their corresponding {alpha}-keto acids. The {alpha}-keto acids are then further converted into aldehydes, alcohols, and esters, which are important aroma compounds. It was shown that branched-chain amino acids, aromatic amino acids, and methionine are catabolized via the transamination route. The elimination route has been described for methionine where activity by carbon-sulfur lyases results in the release of methanethiol.


Figure 2
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  		FIG. 2. Generic amino acid (branched-chain amino acids, aromatic amino acids, and methionine) degradation pathway initiated by transamination in LAB. The enzymes are BcAT, ArAT, AspAT, GDH, HycDH, KdcA, AlcDH, AldDH, and EstA (esterase A). Enzymes specific for branched-chain amino acids degradation (dotted box): KaDH, {alpha}-keto acid dehydrogenase complex; PTA, phosphotransacylase; and ACK, acyl kinase.


Figure 3
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  		FIG. 3. Cysteine and methionine metabolism in LAB. Both biosynthesis and degradation pathways of cysteine and methionine are included in this map, while the catabolic reactions are framed. Biosynthesis of both amino acids comprises a route from serine, a route from aspartate and the interconversion between cysteine and methionine. Degradation of methionine involves the transamination pathway that is initiated by aminotransferases and the elimination pathway initiated by C-S lyases. The enzymes involved in the pathways are CysE, serine acetyltransferase; CysK; CBL; CGL; MGL; CBS; CGS; HSDH; HSK; HSST; HSAT; AHSH; MetH; MetE; AT, aminotransferases; HycDH; and KdcA. The metabolites produced were KMBA, 4-methylthio-2-oxobutanoate; HMBA, 2-hydroxy-4-methylsulfanyl-butyric acid; DMS; DMDS, dimethyl disulfide; and DMTS, dimethyl trisulfide. Key sulfur-containing flavor compounds are colored green-gold.


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ENZYMES INVOLVED IN THE TRANSAMINATION ROUTE
 
Aminotransferases.
Transamination of branched-chain amino acids, aromatic amino acids, and methionine can be catalyzed by different aminotransferases (Fig. 2). In lactococcal strains, branched-chain aminotransferase (BcAT) displays an activity toward both the branched-chain amino acids and methionine (56), while in these strains aromatic aminotransferase (ArAT) is active against aromatic amino acids, leucine, and methionine (55, 56). In Brevibacterium linens, aspartate aminotransferase (AspAT) is responsible for aspartate transamination but is also active on the aromatic amino acids (78). In cheese, flavor compounds such as 2-methylbutanoate (sweaty odor) and isobutyric acid (sour and sweet odor) are believed to be derived from the transamination of Ile or Val by BcAT (6, 77, 78). Table 1 indicates that a BcAT ortholog is present in all lactococcal and streptococcal strains, while it is lacking in a number of lactobacilli such as L. johnsonii, L. sakei, and L. reuteri. Therefore, the latter strains may be unable to produce 2-methylbutanoate and isobutyric acid.


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  		TABLE 1. Distribution of enzymes involved in the transamination route of amino acid degradation in LAB genomese

Genes encoding ArAT and AspAT have been experimentally characterized in L. lactis, e.g., the araT gene from L. lactis NCDO763 (55), and the aspAT gene from L. lactis LM0230 (23). They are found to be homologs, since both belong to the aminotransferase I family (40) (see Fig. S2 in the supplemental material). Putative araT genes were found in all LAB genomes except for L. sakei and L. brevis, while the aspAT gene was absent in LAB species of the L. acidophilus group (Fig. 1).

GDH.
In LAB, the transaminase reaction is commonly linked to the deamination of glutamate to oxoglutaric acid ({alpha}-ketoglutarate), catalyzed by glutamate dehydrogenase (GDH) (Fig. 2). Yvon et al. proposed that the amount of {alpha}-ketoglutarate is the limiting factor for flavor formation from amino acids in cheeses (66, 76). Table 1 shows that gdh genes are only found in the genomes of L. plantarum, L. salivarius, and S. thermophilus strains, which agrees with the strain dependency of the presence of GDH and the lack of GDH activity in most LAB strains (66). However, GDH activity was more commonly found in natural strains used for cheese manufacture (66).

{alpha}-Keto acid conversion enzymes.
{alpha}-Keto acids can be further converted in three different routes (Fig. 2). The {alpha}-keto acid decarboxylase gene (kdcA), for conversion to the corresponding aldehydes, was characterized from L. lactis B1157 by Smit et al. (64). No orthologs were found in the sequenced LAB genomes, except a gene with partial homology in L. lactis subsp. lactis IL1403 (Table 1). This is consistent with the observations that keto acid decarboxylase (KdcA) activity was only found in nondairy lactococcal strains, and only small amounts of amino-acid derived aldehydes were produced by most LAB (62-65). However, alcohols and carboxylic acids that can be derived from those aldehydes were detected in many LAB, suggesting alternative pathways to produce these flavor compounds.

{alpha}-Keto acids can be directly converted to carboxylic acids via oxidative decarboxylation. The first step is the conversion of an {alpha}-keto acid to its corresponding acyl coenzyme A (acyl-CoA) by {alpha}-keto acid dehydrogenase (KaDH), an enzyme complex composed of the four subunits: E1{alpha}, E1β, E2, and E3. Acyl-CoA is further converted to the corresponding carboxylic acid by a phosphotransacylase and acyl kinase (ACK) (Fig. 2). The oxidative decarboxylation pathway was characterized in Enterococcus faecalis (70), and phosphotransacylase, ACK, and a KaDH complex with specific activity toward their corresponding substrates derived from branched-chain amino acids were found encoded in one operon (ptb-buk-bkdDABC). Only the L. casei genome contained a similar orthologous operon. Nevertheless, homologs of the ptb gene, buk gene, and bkdDABC genes were found encoded separately in different positions of the chromosome in various other LAB, for instance in S. thermophilus. This finding agrees well with the experiments of Helinck et al. (37), who showed these enzyme activities in S. thermophilus strains. Caution is required, however, since the best homologs of KaDH in many LAB are annotated as either pyruvate or acetoin dehydrogenase complex, and it is not clear whether these complexes have overlapping substrate specificity.

{alpha}-Keto acids can also be reduced to hydroxy acids by hydroxy acid dehydrogenase (HycDH). Two stereospecific enzymes—D-HycDH and L-HycDH—are distinguished that belong to the larger D-lactate dehydrogenase (D-LDH) and L-LDH protein families, respectively. L-HycDH from Weissella confusa (formerly Lactobacillus confusus) has been characterized, while a D-HycDH encoding gene has been cloned from L. casei (42, 49). Our analysis showed that HycDH enzymes can be clearly distinguished from their closely related LDH homologs (see Fig. S3 in the supplemental material). Table 1 indicates that most LAB possess one or more L-HycDH, while only some have a D-HycDH. Although we could not find any literature evidence that hydroxy acids can directly lead to flavor formation, the shared precursors of hydroxy acids and some flavor compounds imply that the activity of HycDH could have a negative effect on flavor formation by shunting flavor precursors into nonflavor products.

AlcDH and AldDH.
Alcohol dehydrogenase (AlcDH) and aldehyde dehydrogenase (AldDH) catalyze the conversion of aldehydes to alcohols and carboxylic acids, respectively. The neighbor-joining trees of both the AlcDH and the AldDH families appear to be large and complicated (51, 54). Since the experimental evidence of AlcDH and AldDH from LAB is lacking, only several putative AlcDH subfamilies and a bifunctional AlcDH/AldDH subfamily can be distinguished (data not shown). Table 1 shows the genes encoding putative AlcDH and bifunctional AlcDH/AldDH proteins with AldDH catalytic domains. Most LAB genomes encode multiple AlcDH members, but only a single AlcDH/AldDH ortholog.

Esterases.
Esters with short-chain fatty acids as the precursors, such as ethyl butanoate and ethyl isovalerate, can be formed via amino acid degradation pathways (Fig. 2). They are important for development of the characteristic "fruity" flavor notes in cheeses. The gene estA encoding an esterase that can catalyze the biosynthesis of esters derived from short-chain fatty acids was cloned and characterized in lactococcal strains (28), as well as in Lactobacillus helveticus (26) and L. casei (74). These estA genes shared high sequence similarity and could be easily distinguished from other esterases (17, 26; data not shown). Many LAB genomes, like those of the lactococci and streptococci, have one estA gene (Table 1). A study of an esterase-negative mutant of L. lactis confirmed that the estA encodes the only enzyme for the synthesis of short-chain fatty acid esters in this species (48). Therefore, the absence of the estA gene in L. acidophilus, L. johnsonii, L. salivarius, and other species (Table 1) could imply that these strains do not synthesize short-chain fatty acid esters.


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ENZYMES INVOLVED IN FLAVOR FORMATION FROM METHIONINE/CYSTEINE METABOLISM
 
Methanethiol and other key sulfuric aroma compounds such as dimethyl sulfide (DMS), dimethyl disulfide, and dimethyl trisulfide have a significant impact on cheese sensory profiles (72). They are normally derived from methionine and cysteine. Since methionine and cysteine are usually present in only limited amounts in the milk environment, either as free amino acids or in milk proteins (52, 58), the formation of sulfur-containing flavor compounds will depend on both biosynthesis and catabolic pathways of methionine and cysteine. Figure 3 gives an overview of the metabolic pathways involved. Hydrogen sulfide can be released from cysteine or homocysteine by the activities of several C-S lyases or cysteine synthase (CysK) (Fig. 3), which leads to increased H2S levels during cheese ripening (7, 34, 46). Several chemical reactions can also contribute to the sulfuric flavor compounds formation, e.g., converting cystathionine to DMS or liberating methanethiol from methionine in the presence of pyridoxal phosphate (PLP) (72, 73).

The metabolism of sulfur-containing amino acids is complex, especially considering the existence of multiple alternative pathways/enzymes for methionine biosynthesis (36). Our genome-wide analysis of LAB showed large differences in the distribution of the related enzymes, indicating a high variability in the presence of the different routes (Table 2). Compared to L. plantarum and S. thermophilus strains, in which most of these genes seem to be present, the other LAB genomes lack many of these enzymes. Most S. thermophilus strains exhibit no absolute amino acid requirements for growth on minimal medium, including sulfur-containing amino acids (43), which agrees with our prediction that all biosynthesis enzymes are present. The distribution of these enzymes can also vary between different strains of the same LAB species. For instance, in the L. delbrueckii subsp. bulgaricus ATCC 11842 genome, a metE gene is absent and two homoserine O-succinyltransferase (HSST)-encoding genes (metA) are inactivated due to point mutations, while those genes are present in L. delbrueckii subsp. bulgaricus ATCC BAA365. Similarly, one of the cbl/cgl genes is truncated in L. lactis subsp. cremoris MG1363, and cgs and metA appear to be pseudogenes in L. lactis subsp. cremoris SK11. It should be noted that these three genes are located in the same operon in Lactococcus genomes, indicating putative frequent mutation events in this region leading to gene inactivation (see Fig. S4 in the supplemental material).


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  		TABLE 2. Distribution of genes encoding enzymes involved in methionine and cysteine metabolism in LAB genomese

Homoserine activation enzymes.
Methionine biosynthesis from homoserine can be initiated by one of three reactions: a homoserine kinase (HSK) reaction, a homoserine trans-succinylase reaction, and a homoserine trans-acetylase reaction, which are catalyzed by HSK, HSST, and homoserine O-acetyltransferase (HSAT), respectively (Fig. 3). No homolog of HSAT from Bacillus cereus was found using BLAST in any of the sequenced LAB genomes, whereas HSK and HSST are distributed relatively broadly in LAB. Although HSST and HSAT belong to different protein families and share little sequence similarity, previous studies revealed that they share overlapping substrate specificity (36). In Bacillus subtilis, HSST is likely to possess homoserine trans-acetylase activity (14, 57). Moreover, the orthologous gene in L. plantarum is located in the same operon as the genes encoding O-acetylhomoserine sulfhydrylase (AHSH) and homoserine dehydrogenase (HSDH), as in Bacillus (see Fig. S4 in the supplemental material). The genetic organization reflects the functional correlation between HSDH, HSST, and AHSH in L. plantarum, a bacterium that is known to lack a tricarboxylic acid cycle to make succinyl-CoA and therefore is restricted to using acetyl-CoA to synthesize methionine (33). Thus, we hypothesize that HSAT activities can be complemented by HSST in some LAB and that HSST, together with AHSH, can form homocysteine, the precursor of methionine.

Homocysteine methylation enzymes.
Methylation of homocysteine, the final step of methionine biosynthesis, can be catalyzed by either of two nonhomologous enzymes, i.e., a cobalamin-dependent homocysteine methyltransferase (MetH) or a cobalamin-independent methionine synthase (MetE). Both MetH and MetE are present in L. reuteri strains, S. thermophilus strains, and L. delbrueckii subsp. bulgaricus strain ATCC BAA365, whereas in some other LAB genomes only one enzyme is found (Table 2).

Lyases and synthases: the CysK/CBS family.
CysK (cysteine synthase or O-acetylserine-thiol-lyase) and CBS (cystathionine beta-synthase) belong to the same protein family, the so-called pyridoxal phosphate-dependent β-family (18, 38, 47). Five major subfamilies could be distinguished in the phylogenetic tree of the CysK/CBS protein family with relatively good bootstrap support (Fig. 4). This protein family includes the tryptophan synthase β-subunit, threonine dehydratase, and threonine synthase subfamilies. The CysK and CBS subfamilies are more closely related to each other than to the other three subfamilies. As a result, the attribution of substrate specificities of these enzymes was less straightforward. In addition, the only characterized cysK gene in LAB is from L. lactis subsp. cremoris MG1363 (30), while none of cbs gene products have been studied in any LAB. Actually, all of the putative enzymes belonging to this CysK/CBS cluster were originally annotated as cysteine synthase (CysK) in LAB genomes and given genes names cysK or cysM (see Table S1 in the supplemental material). A recent study on the enzymes converting methionine to cysteine in B. subtilis (38) has found that both CysK and CBS (formerly named YrhA in B. subtilis) have an O-acetylserine-thiol-lyase (cysteine synthase) activity, although CysK represented 95% of this activity. In addition, an atypical cystathionine β-synthase activity was observed for CBS but not for CysK in B. subtilis.


Figure 4
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  		FIG. 4. Phylogenetic tree of the CysK/CBS family. Different colors represent orthologous groups. Red squares represent duplication events, while green circles represent speciation events. Bootstrap values are reported for a total of 1,000 replicates. Red dots indicate the experimentally verified genes from L. lactis subsp. cremoris MG1363 (LLM_CysK), B. subtilis (BSU_CysK and BSU_yrhA_CBS), and F. nucleatum (FN_CysK). The subfamilies related to the tryptophan synthase β-subunit, threonine dehydratase, and threonine synthase could be distinguished via analysis of the gene context, since most of the corresponding genes are associated with specific amino acid biosynthesis gene clusters (data not shown). The GI code of each protein in the family is preceded by its corresponding genome abbreviation.

The information on cysK and cbs obtained from B. subtilis and on cysK obtained from Fusobacterium nucleatum was added to Fig. 4 to increase the specificity of the functional annotation of the CysK/CBS cluster (34). The branches with the CysK sequences from B. subtilis, L. lactis, and F. nucleatum contain orthologous sequences from all lactococcal and streptococcal strains, as well as from several Lactobacillus strains, mainly from the L. acidophilus group. As Fig. 4 shows, a relatively recent duplication in the CysK subfamily has occurred in lactococcal genomes. In L. lactis subsp. cremoris MG1363, one of the paralogs was found to be cotranscribed with the gene encoding cystathionine beta/gamma lyase (which belongs to CBL/CGL lyase group [described below]) (see Fig. S5 in the supplemental material), and to be under the regulation of the LysR-type regulator CmbR (29, 35). In contrast to CysK, a protein orthologous to B. subtilis CBS (YrhA) seems to be absent in lactococcal genomes. This could cause a slower growth of lactococci utilizing homocysteine as the only sulfur source, since this phenotype is observed for a cbs (yrhA) mutant from B. subtilis (38). Our subdivision into CysK and CBS protein subfamilies was strengthened by the observation that almost all of the putative cbs orthologs have the same gene context, being located in the same operon, together with a cbl/cgl gene (belongs to CBL/CGL lyase group [discussed below]). In fact, the genetic organization is similar to that of the metC-cysK operon in lactococci (see Fig. S5 in the supplemental material).

Lyases and synthases: the CBL/CGL families.
Methionine elimination is a major pathway of methionine degradation in some cheese microorganisms (78). This pathway is initiated by the dethiomethylation reaction, in which methionine is converted to methanethiol by C-S lyases, including methionine {gamma}-lyase (MGL), cystathionine β-lyase (CBL), and cystathionine {gamma}-lyase (CGL). MGL catalyzes the {alpha},{gamma}-elimination of methionine, and this activity is observed mainly in brevibacteria and corynebacteria (27). The mgl gene from B. linens was sequenced, and gene disruption has shown its essential role for flavor formation (4). However, none of the LAB genomes seem to have the mgl gene, which is in agreement with the near absence of the related enzyme activity observed in LAB (21).

CBL and CGL catalyze an {alpha},β-elimination and an {alpha},{gamma}-elimination reaction, respectively, converting cystathionine to homocysteine or cysteine (Fig. 3). Moreover, it was shown that the enzymes are capable of converting other sulfur-containing substrates via {alpha},{gamma}-elimination. For instance, methanethiol is formed from methionine, although the enzyme activity toward methionine was approximately 10- to 100-fold lower than toward cystathionine (72, 78). CBL has been purified from various LAB, and its gene was previously identified from L. lactis (called metC) and L. debrueckii subsp. bulgaricus (called patC) (2, 7, 29). CGL enzyme activity has been detected in L. reuteri (20), L. fermentum (61), and L. lactis subsp. cremoris SK11 (13), while the gene from L. lactis subsp. cremoris MG1363 was characterized experimentally (22). Analysis of substrate specificities suggests an overlapping function between CBL and CGL. For example, it has been reported that the gene encoding CBL from L. lactis subsp. cremoris B78 or MG1363 is also capable of catalyzing the {alpha},{gamma}-elimination reaction like CGL (2, 22).

Sequence analysis of the functional domains of experimentally verified cbl and cgl gene products revealed that they fall into two distinct families, which share little sequence similarity. The first family is the aminotransferase I protein family, which mainly consists of various aminotransferases, including the previously mentioned ArAT and AspAT (see Fig. S2 in the supplemental material). We propose to use cblA/cglA as names for the genes encoding CBL/CGL in this aminotransferase I family. Three experimentally characterized genes encoding CBL or CGL were found to belong to this family: the patC gene of L. debrueckii subsp. bulgaricus (7), the ytjE gene from L. lactis subsp. lactis IL1403 (46), and the malY gene from L. casei (39). All of these enzymes exhibit {alpha},β-elimination activity toward cystathionine; however, the one from L. lactis can also catalyze {alpha},{gamma}-elimination of methionine. Orthologs of the cblA/cglA genes from the aminotransferase family are found in many LAB genomes (Table 2).

The second family to which CBL/CGL enzymes belong has been designated as the lyase family and includes various methionine or cysteine metabolism-related enzymes: CBL, CGL, and cystathionine {gamma}-synthase (CGS), as well as O-acetyl homoserine sulfhydrylase (AHSH). The neighbor-joining tree of this family clearly shows three major subfamilies: the CBL/CGL subfamily, the CGS/AHSH subfamily, and the AHSH subfamily (Fig. 5). The first major subfamily can be divided into two functional clusters named "CBL/CGL" and "CBL," respectively, with the proposed gene names cblB/cglB. They can be distinguished by including experimentally characterized CBL/CGL proteins, encoded by the metC genes from L. lactis subsp. cremoris MG1363 and B78 (22, 30), the yrhB gene (renamed cgl in Fig. 5) (38), and the metC gene (formally yjcJ, renamed cbl in Fig. 5) from B. subtilis (8). The CGL and CBL from B. subtilis could be separated into two subclusters, representing different substrate specificities and physiological roles. Experimentally, the B. subtilis CGL enzyme was found to possess CGL activity but was not able to degrade methionine (38). On the other hand, CBL activity was detected from the CBL enzyme in B. subtilis, but its activity toward methionine was not determined (8). The metC genes from L. lactis subsp. cremoris grouped together with CGL from B. subtilis. However, unlike the B. subtilis CGL, the enzymes encoded by the lactococcal metC genes were found to carry out both {alpha},β-elimination and {alpha},{gamma}-elimination toward different substrates (22). Therefore, the subgroup "CBL/CGL" seems to display a mixture of cystathionine beta- and gamma-lyases activities. It implies that LAB enzymes in this subcluster could have either solo CGL activity or a dual CBL/CGL activity.


Figure 5
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  		FIG. 5. Neighbor-joining tree of the CBL/CGL lyase family. Different colors represent orthologous groups. Red squares represent duplication events, while green circles represent speciation events. Bootstrap values were calculated for a total of 1,000 replicates. Experimentally characterized proteins are indicated by red dots, including CGL, CBL, and CGS from B. subtilis (BSU_yrhB_CGL, BSU_yjcJ_CBL and BSU_yjcI_CGS, respectively), the CBL/CGL from L. lactis subsp. cremoris MG1363 and NIZO B78 (LLC1_MetC and LLC2_MetC, respectively), the MetB from L. casei (LCA_MetB), and the CGS from S. anginosus (SAN_CGS), as well as the AHSH (cysD gene) from E. nidulans (NAI_CysD). The GI codes of the other LAB proteins in this family are shown after the genome abbreviation.

The second major subfamily in the lyase family contains the yjcI gene (renamed cgs in Fig. 5) from B. subtilis and the cgs gene from Streptococcus anginosus. Both genes encode bifunctional enzymes which have both CGS and AHSH activities (8, 75). Thus, we define this protein subfamily as the (bifunctional) "CGS/AHSH" subfamily, and assumed that all of the members may exhibit both CGS and AHSH activities.

The third major subfamily contains unifunctional AHSH enzymes, as suggested by the experimentally characterized cysD gene from the fungus Emericella nidulans and other microorganisms (15, 59, 60). Notably, almost all of the LAB species/strains that contain a member of the AHSH subfamily also seem to contain a CGS/AHSH subfamily member, except for Oenococcus oeni. Unfortunately, to our knowledge, no experimental evidence on the characterization of CGS or AHSH from LAB is available.


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IMPROVED ANNOTATION AND NOMENCLATURE
 
In this review, we focused on the enzymes involved in flavor-forming pathways, especially the ones which catalyze the metabolism of sulfur-containing amino acids, leading to sulfuric flavor compounds. By combining phylogenetic analysis, experimental evidence, and gene context analysis, we could improve the original annotations of many of the genes encoding these enzymes in LAB. Some of the previous annotations were sometimes inconsistent and caused much confusion. A more consistent nomenclature for genes and proteins is proposed (Tables 1 and 2 and see Table S1 in the supplemental material). Various enzymes belong to large protein families. For instance, aminotransferases ArAT, AspAT, and cystathionine lyases CBL/CGL fall into the same aminotransferase family I. Subfamilies with different substrate specificities could be distinguished by our method. Some of the enzymes exhibit overlapping substrate specificities, e.g., ArAT, AspAT, and BcAT, although they are still expected to have a preference toward certain substrates. The cbs genes from LAB, some of which were previously annotated as cysteine synthase (CysK), are now for first time proposed to be cystathionine β-synthase instead. Finally, some enzymes are predicted to be able to catalyze different types of reactions, for example, proteins from the CBL/CGL subfamily may carry out both {alpha},β-elimination and {alpha},{gamma}-elimination reactions, and proteins from CGS/AHSH subfamily might display both CGS and AHSH activity.


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FLAVOR-FORMING POTENTIAL
 
The improved gene annotation leads to a better prediction of the flavor-forming potential. S. thermophilus and Lactococcus strains, as well as some other milk-associated LAB, e.g., L. casei, seem to possess relatively more abundant genes encoding flavor-related enzymes, whereas many of these enzymes are lacking in L. gasseri and L. johnsonii, which are typical bacteria living in the gastrointestinal tract. The L. plantarum genome also encodes a large set of enzymes involved in sulfur-containing amino acid metabolism, which might reflect its flexibility to grow under different conditions (41). It is important to keep in mind that only the LAB type strains, which have been completely sequenced, were analyzed in the present study. It has been shown that in some cases the presence of flavor-forming enzymes can vary between strains from the same species. Therefore, in order to explore the flavor-forming ability of other LAB strains, which have not been sequenced, especially the ones for industrial use, experimental techniques such as CGH microarrays (i.e., comparative genomic hybridization with microarray) could be applied. On the other hand, high-throughput genome sequencing is now becoming so fast and affordable that numerous industrial and environmental LAB strains can soon be sequenced. The importance of our in silico analysis is in guiding future genomics sequencing and experimentation and thereby validating our predictions.

In conclusion, the identification of key enzymes in flavor forming pathways and the prediction on flavor-forming capacity of various LAB should provide an excellent starting point to direct the selection of potential species and/or strains used for the industrial production of flavored products.


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ACKNOWLEDGMENTS
 
This study was supported by grant CSI4017 from the Casimir program of the Ministry of Economic Affairs, The Netherlands. C.F. is supported by the Kluyver Center for Genomics of Industrial Fermentation.

We thank Johan van Hylckama Vlieg for critically reading the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: (31)24-3619827. Fax: (31)24-3619395. E-mail: m.liu{at}cmbi.ru.nl Back

{triangledown} Published ahead of print on 6 June 2008. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, August 2008, p. 4590-4600, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.00150-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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