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Applied and Environmental Microbiology, April 2009, p. 2326-2332, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02417-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Heterologous Production of Methionine--Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds ^,

Sean B. Hanniffy,¹ Mark Philo,¹ Carmen Peláez,² Michael J. Gasson,¹ Teresa Requena,² and M. C. Martínez-Cuesta^2*

Food Safety Science Division, Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UA, United Kingdom,¹ Department of Dairy Science and Technology, Instituto del Frío (CSIC), José Antonio Novais 10, 28420 Madrid, Spain²

Received 21 October 2008/ Accepted 16 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine--lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

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Methionine (Met) catabolism plays a major role in cheese flavor development. Met is believed to be the precursor of numerous diverse and quantitatively minor volatile sulfur compounds (VSCs) (38) which make important contributions to the overall flavor that are typical to different cheeses (7). Most of these compounds are derived from the degradation of Met to methanethiol (MTL), giving rise to a variety of compounds such as dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters (41).

The Met biosynthetic and catabolic pathways leading to MTL vary among bacteria (36), as do the enzymes involved and the amount of MTL produced during cheese ripening (15). The most direct pathway occurs via L-methionine -elimination of Met to MTL, -ketobutyrate, and ammonia. This L-methionine -elimination activity has been shown to be quite efficient in Brevibacterium linens (21), while its presence has been suggested in several other cheese surface bacteria such as Micrococcus luteus, Arthrobacter sp., Corynebacterium glutamicum, and Staphylococcus equorum (9). In B. linens, this activity is catalyzed by an L-methionine--lyase (MGL), which has been previously purified and characterized (16). Moreover, disruption of the mgl gene encoding the enzyme has been shown to almost eliminate this strain's considerable capacity to produce VSCs (2). In lactococci, which produce only limited amounts of VSCs (15), this reaction is catalyzed by cystathionine lyases (β- or -) which are responsible for the simultaneous deamination and demethylthiolation of Met to MTL (1, 11, 18, 22); recently, a new C-S lyase (YtjE) with ,-elimination activity that degrades Met into MTL has been characterized in our laboratory (30). Unfortunately, C-S lyases display relatively low activities toward Met, limiting their capacities to produce VSCs. Another route for Met catabolism involving a two-step mechanism initiated by an aminotransferase has also been identified in lactococci (8, 23) and other cheese-ripening bacteria (3, 9), which leads to the formation of -keto--methylthiobutyric acid which is subsequently converted to MTL; this route, however, produces only limited amounts of MTL.

In recent years, numerous studies have pursued the control and/or diversification of VSCs primarily by means of using selected cheese-ripening microorganisms or combinations of them (4, 7, 25). A few studies have focused on engineering lactic acid bacteria (LAB) with enhanced VSC-producing abilities by increasing cystathionine lyase activities (22, 27). In this respect, a Lactococcus lactis strain engineered to overproduce cystathionine β-lyase was shown to produce larger quantities of VSCs with Met as a substrate compared to the wild-type strain (22). However, this study also showed no significant difference in VSC production between the wild-type strain and a cystathionine β-lyase-knockout variant, implying that other enzymes may play a more prominent role in the conversion of Met.

The aim of this study was to develop LAB with improved capability to produce MTL and other related VSCs as a more efficient strategy to enhance cheese flavor development. We describe the bioengineering of food-grade L. lactis strains to produce MGL, which has been reported to play a major role in the catabolism of Met to MTL in B. linens, a good producer of VSCs (2). The enzyme activity of the recombinant MGL was confirmed, and its role in the production of VSCs by recombinant L. lactis was also investigated.

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Bacterial strains and culture conditions.
L. lactis strains MG1363 (24) and FI7847 nisA (19), used for cloning and expression of recombinant protein, respectively, were grown at 30°C without agitation in M17 medium supplemented with 0.5% (wt/vol) glucose (G-M17). Plasmids were electroporated into L. lactis (42), and transformants were selected on G-M17 agar (1.5%) plates supplemented with erythromycin (5 µg ml^–1) (Sigma, Dorset, United Kingdom) to select for recombinant pTnis and pTnis-His plasmids (32, 33). B. linens strain CECT 76 (Spanish Type Culture Collection) was grown at 25°C with aeration (250 rpm) in Trypticase soy broth. Escherichia coli BL21(DE3) expressing YtjE (30) was grown aerobically at 37°C in Luria-Bertani medium supplemented with kanamycin (30 µg ml^–1) (Sigma). Growth of bacterial cultures was routinely monitored by measuring the optical density at 600 nm (OD₆₀₀).

DNA manipulations.
Molecular cloning techniques were performed essentially as previously described (34). Plasmid DNA from L. lactis was isolated using the Qiagen plasmid miniprep procedure (Qiagen, Ltd., Crawley, United Kingdom) adapted to include mutanolysin (0.1 U µl^–1) and lysozyme (5 mg ml^–1) in the resuspension buffer to facilitate cell wall degradation. Extraction and purification of the plasmid DNA were performed using the MiniElute Qiagen kit. Vent DNA polymerase (New England Biolabs [NEB], Herts, United Kingdom) was used for PCR amplification reactions according to the manufacturer's instructions. DNA restriction and modification enzymes were purchased from Promega (Southampton, United Kingdom). DNA sequencing was carried out as a service at Lark Technologies, Inc. (Takeley, Essex, United Kingdom).

Codon usage optimization of the mgl gene sequence of B. linens and cloning and expression in L. lactis.
The pTnis plasmid, a derivative of the gram-positive, broad-host-range vector pTREX1 (32, 33), was used to express MGL in L. lactis. The gene encoding MGL from B. linens (GenBank/DDBJ/EMBL accession no. AY622198) (2) was synthetically manufactured by Epoch Pharmaceuticals. To enhance the translational efficiency (i.e., the expression level of heterologous protein), we first codon optimized MGL by adjusting the codon frequency to that of L. lactis MG1363 (40) (see the supplemental material). Using the synthetic mgl gene as a template, the Mgl forward (5'-CATGCATGCAGATTACCCAAAATGGAATTTCAAC-3') and Mgl reverse (5'-CGGGATCCTTAAACAGTTGCAACTGGATG-3') oligonucleotide primers (adapted to include the restriction sites SphI and BamHI) were used to PCR amplify an mgl gene fragment to facilitate cloning into pTnis, downstream of a nisin-inducible promoter (14). A second mgl gene fragment was also PCR amplified using the oligonucleotide primers Mgl Forward (see above) and Mgl reverse2 (5'-CCCTCGAGAACAGTTGCAACTGGATGTTG- 3'), the latter adapted to include an XhoI site to facilitate cloning into the plasmid pTnis-His, in frame with a carboxyl-terminal His₆ tag (encoded by the vector) to enable downstream purification of recombinant protein. The cloned mgl gene was identical to the codon-optimized gene, except for the changing of a triplet of bases (CAG for AGT) that resulted in the introduction of the amino acid Gly instead of Ser (at position 2) at the N terminus of the recombinant MGL protein. The PCR-amplified products (representing mgl and mgl-HT) were digested and then ligated into appropriately digested pTnis and pTnis-His plasmid vectors. Recombinant plasmids were then transformed into L. lactis MG1363. Plasmid DNA was then prepared from individual clones and analyzed by DNA sequencing. Plasmid DNA from clones with the correct DNA sequence were then transformed into the expression host L. lactis FI7847 nisA (19) to generate L. lactis pTnis-mgl and L. lactis pTnis-mgl-HT.

Purification of recombinant MGL and YtjE proteins.
To optimize MGL expression, L. lactis FI7847 nisA cultures containing pTnis-mgl-HT were grown at 30°C to an OD₆₀₀ of 0.5 before induction with nisin (1 to 50 ng ml^–1) for 3 h. To purify recombinant MGL (His tagged), the cultures were induced with 25 ng ml^–1 of nisin for 3 h at 30°C, at which point the pellets were collected by centrifugation and stored at –20°C. Purification of His-tagged MGL was performed as previously described (35). Briefly, cell pellets were resuspended in 10 ml of lysis buffer (50 mM sodium phosphate [pH 7.5], 300 mM NaCl, 10 mM imidazole, 10% glycerol) containing 1 mM EDTA, 1 mg ml^–1 of lysozyme, 1 mM phenylmethylsulfonyl fluoride, 100 µM pyridoxal 5'-phosphate (PLP) and an EDTA-free protease inhibitor cocktail tablet (Roche). Cell suspensions were incubated for 30 min on ice, disrupted by glass beads, and centrifuged (12,000 x g, 20 min) at 4°C to remove insoluble materials. The clear supernatant was mixed with 1.5 ml of a 50% slurry of Ni²⁺ and nitrilotriacetic acid resin (Qiagen) at 4°C under gentle shaking for 4 h before being loaded into a polypropylene column. After unbound proteins were removed through a series of washes, recombinant protein was eluted in elution buffer (50 sodium phosphate [pH 7.5], 300 mM NaCl, 300 mM imidazole, 10% glycerol). Eluted MGL was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and selected fractions were pooled and dialyzed against phosphate-buffered saline (pH 7.0) solution containing 10% glycerol. The YtjE protein, a C-S lyase used for comparative analyses, was expressed and purified as described previously (30). Recombinant proteins were quantified using the Bradford assay method (10).

Preparation of cell samples for enzymatic and VSC production analyses.
Recombinant MGL activity was assayed using either purified His-tagged MGL protein, whole cells, or cell extracts (CFEs) prepared from nisin-induced cultures of the L. lactis pTnis-mgl strain. Whole cells and CFEs prepared from cultures of L. lactis FI7847 nisA carrying empty vector (nisin induced) and B. linens were used as controls. Cells from L. lactis cultures grown to an OD₆₀₀ of 0.5 before nisin induction for 3 h and B. linens cultures (50 ml) grown to an OD₆₀₀ of 1.2 were harvested by centrifugation (3,220 x g, 15 min at 4°C), washed twice in an equal volume of ice-cold potassium phosphate buffer (50 mM; pH 7.0) and adjusted to an OD₆₀₀ of 20 in 100 mM Tris HCl (pH 8.0) or 50 mM sodium citrate (pH 5.4; 4.5% NaCl); VSC analyses were performed immediately with these bacterial suspensions. To prepare CFEs, cell pellets obtained as explained above were resuspended in 2 ml of ice-cold cell suspension buffer (50 mM potassium phosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 100 µM PLP [pH 7.5]). A 1-ml aliquot was then transferred to a microcentrifuge tube containing 1 g of sterile glass beads (1-mm diameter) (Sigma) and vortexed (four times for 2 min with 1-min intervals on ice). The insoluble fraction and beads were removed by centrifugation (10,000 x g) for 10 min at 4°C, and the supernatant fraction containing soluble protein was aliquoted, frozen in liquid nitrogen, and stored at –80°C until used in enzyme assays.

Enzyme assays.
The likely C-S lyase activity of recombinant MGL was determined by measuring the formation of free thiol groups according to a method previously described (39) and using L-cystathionine, L-cystine, or L-methionine as the substrate. Reaction mixtures containing 0.2 mM [5,5'-dithiobis (2-nitrobenzoic acid)] (DTNB), 100 µM PLP, substrate (at different concentrations), and either His-tagged MGL or CFEs suspended in 100 mM Tris HCl (pH 8.0) solution, were incubated at 37°C for 1 h, and the increase in A₄₁₂ was measured at 2-min intervals. Reactions were also performed under cheese-ripening conditions (50 mM sodium citrate solution [pH 5.4], 4.5% NaCl). Control assays were performed in which no CFEs or protein was added in order to subtract values associated with nonspecific reactions. Purified YtjE, a C-S lyase from L. lactis that degrades L-methionine (30), was used as a control. The molar absorption coefficient value used for aryl mercaptide was 13,200 liters mol^–1 cm^–1, with 1 enzyme unit representing the formation of 1 µmol aryl mercaptan min^–1.

Detection of C-S lyase activity by in situ staining.
The activity of recombinant MGL toward L-cysteine (L-cysteine desulfhydrase activity) was assayed by visualizing enzyme activities (5, 43, 44). The activities of purified His-tagged MGL or CFEs from recombinant L. lactis cultures were monitored using Tris-glycine gels (Invitrogen) run under nondenaturing conditions. Purified YtjE and CFEs prepared from B. linens and L. lactis FI7847 nisA carrying empty vector were included for control purposes. Following electrophoresis (20 mA at 4°C for 2 h), gels were incubated at 37°C in 10 ml of the visualizing solution containing 100 mM triethanolamine HCl (pH 7.6), 100 µM PLP, 0.5 mM bismuth trichloride, 10 mM EDTA, 1% Triton X-100, and 5 mM L-cysteine to detect a black precipitate at the position of enzyme activity.

VSCs produced by recombinant MGL.
Recombinant MGL activity toward L-methionine was also determined by analyzing the production of VSCs. Reactions were carried out both in 100 mM Tris HCl (pH 8.0) and in 50 mM sodium citrate (pH 5.4, 4.5% NaCl) (cheese-ripening conditions) containing 100 µM PLP and either 5 mM L-methionine mixed with purified His-tagged MGL protein, whole cells, or CFEs from recombinant L. lactis cultures and incubated at 30°C for 48 h (His-tagged MGL protein and CFEs) and 120 h (whole cells). YtjE protein, whole cells, and CFEs prepared from cultures of L. lactis FI7847 nisA carrying empty vector (nisin induced) and B. linens were also included for comparison. The products formed were determined by gas chromatography-mass spectrometry (GC-MS) as described previously (30). Areas of peaks were calculated using the Chem-station software (Agilent Technologies UK, Ltd.), selecting the peaks manually. Sulfur-containing compounds were identified by comparing retention time and mass spectra with those obtained using standards prepared individually or as mixtures using pure stocks of MTL, DMDS, and DMTS (Sigma).

Chemicals.
Unless otherwise noted, all chemicals were purchased from Sigma (Poole, Dorset, United Kingdom).

Nucleotide sequence accession number.
The mgl codon-optimized nucleotide sequence reported in this paper was submitted to the GenBank database and assigned accession no. FJ542125.

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Cloning and heterologous expression of the B. linens MGL protein in L. lactis. (i) Purification of MGL.
In the first approach, the 1,277-bp DNA fragment carrying the native mgl gene was amplified from B. linens BL2 genomic DNA by PCR using the forward (5'-GGCATGCAAAGTATCACCCAGAACGGAATCTC-3') and reverse (5'-GGGATCCTCATACCGTTGCTACAGGGTG-3') primers, to be cloned into the pTnis plasmid vector in order to overexpress in L. lactis. However, and despite several attempts, we were unable to stably clone the native mgl gene from B. linens into L. lactis as it was prone to mutation (intermolecular recombination events and/or segmental deletions), possibly on account of its high GC content. As a second approach and with a view toward also increasing translation effciency, the mgl gene sequence was codon optimized according to the codon adaptation index previously determined for the L. lactis MG1363 strain and generated as a synthetic DNA fragment as described in Materials and Methods. The codon-optimized mgl gene was subsequently successfully cloned into pTnis and pTnis-His plasmid vectors and expressed as recombinant protein in L. lactis (the latter as a C-terminal His-tagged fusion protein). SDS-PAGE analysis of nisin-induced CFEs of the recombinant L. lactis strains confirmed expression of MGL, which was detected in both the soluble and insoluble fractions (results not shown). The L. lactis pTnis-mgl-HT strain was used to produce recombinant protein (MGL-HT) in order to be purified for further analysis. The L. lactis pTnis-mgl strain was used in enzymatic and VSC determination assays. L. lactis pTnis carrying empty vector was also included in the assays as a control.

His-tagged MGL protein was purified under native conditions by affinity chromatography on nickel-nitrilotriacetic acid resin, and its purity was confirmed by SDS-PAGE. The results identified MGL as a protein of approximately 47 kDa, in agreement with the molecular mass predicted using its deduced amino acid sequence (45.7 kDa).

(ii) Characterization of the recombinant MGL activity.
While the activity of recombinant MGL expressed in L. lactis was characterized primarily by its ability to convert L-methionine into VSCs, its ability to degrade other sulfur-containing compounds was also examined. In this respect, the ability to degrade L-methionine, L-cystathionine, and L-cystine was determined by measuring the formation of free thiol groups (using purified recombinant His-tagged MGL or CFEs prepared from recombinant L. lactis pTnis-mgl). When CFEs were tested under optimum assay conditions (pH 8.0), the formation of free thiol groups was higher (Table 1) in nisin-induced cultures of L. lactis pTnis-mgl than that in the vector control strain, indicating a role for MGL in the degradation of these substrates. Furthermore, activity toward each substrate was shown to correlate with increasing levels of nisin (and thereby MGL expression), reaching optimal levels when cultures were induced with 25 ng ml^–1 of nisin (Table 1). At this concentration of nisin, recombinant MGL activity was comparable to that obtained using CFEs from B. linens. When tested under conditions that mimic cheese ripening, CFEs from recombinant L. lactis pTnis-mgl retained over 30% of its activity measured under optimum conditions (data not shown). In addition, under optimum conditions, the purified His-tagged MGL protein was shown to demonstrate activities of 357 ± 7 mU/mg for L-cystathionine, 604 ± 15 mU/mg for L-cystine, and 488 ± 13 mU/mg for L-methionine, respectively. When these figures were compared with those obtained using purified recombinant YtjE protein (data not shown), a cystathionine lyase from L. lactis (30), YjtE was shown to exhibit much higher activities toward L-cystine and L-cystathionine; nevertheless, MGL produced in L. lactis demonstrated markedly higher (10 times) specificities toward L-methionine.

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TABLE 1. Enzyme activities toward different sulfur-containing compounds with and without nisin induction

The cysteine desulfhydrase activity of MGL was conveniently monitored in nondenaturing gels using L-cysteine as the substrate. Hydrogen sulfide (H₂S) reacts with bismuth to produce an insoluble product which forms brown to black bands on the gels (43). Purified YtjE protein, which displays activity toward L-cysteine was used as a positive control. Following electrophoresis under nondenaturing conditions, purified His-tagged MGL produced a single strongly reacting band of approximately 170 kDa (Fig. 1), confirming the ability of MGL to catalyze the ,β-elimination reaction of L-cysteine to H₂S. Furthermore, a similar band was also apparent in CFEs prepared from B. linens cultures. Critically, this band could be detected in CFEs prepared from nisin-induced L. lactis cultures expressing MGL. By comparison, this band was absent in CFEs from noninduced recombinant L. lactis pTnis-mgl cultures and CFEs from induced and noninduced cultures of L. lactis carrying vector only (Fig. 1). A smaller band exhibiting activity toward L-cysteine could also be detected in all of the CFEs prepared from L. lactis cultures (Fig. 1).

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FIG. 1. Cysteine desulfhydrase activity by in situ staining. MGL activity was monitored in Tris-glycine gels under nondenaturing conditions using L-cysteine as the substrate. Lanes: M, molecular mass markers; 1, purified recombinant His-tagged MGL protein; 2, purified recombinant YtjE protein; 3, CFEs from B. linens cultures; 4 and 5, CFEs from noninduced (lane 4) and nisin-induced (lane 5) L. lactis pTnis (empty vector); and 6 and 7, CFEs from noninduced (lane 6) and nisin-induced (lane 7) recombinant L. lactis pTnis-mgl.

(iii) Formation of VSCs.
MGL activity toward L-methionine was assayed by GC-MS in order to identify the VSCs formed. The assays were performed under optimum conditions (pH 8.0) using purified MGL or CFEs prepared from recombinant L. lactis strains. As shown in Fig. 2a, MTL and its degradation products, DMDS and DMTS, were readily apparent in reaction mixtures containing purified recombinant His-tagged MGL. These compounds could not be detected in control samples incubated in the absence of recombinant protein, confirming MGL ,-elimination activity toward L-methionine. Furthermore, compared to YtjE, reaction mixtures containing MGL produced at least a 25 times increase in VSC formation indicative of the higher specificity of MGL from B. linens toward L-methionine (Fig. 2a). When CFEs were tested, L. lactis pTnis-mgl produced markedly higher levels of VSCs than those obtained with L. lactis carrying empty vector (Fig. 2b). Production of MTL (in assays carried out with purified protein or CFEs) increased with incubation time along with the formation of DMDS and DMTS. While MTL is expected to be the major reaction product of L-methionine catabolism, DMDS was, by far, the most dominant VSC produced in assays carried out using purified protein (Fig. 2a). This is not surprising as MTL is a highly reactive sulfur compound that quickly reacts with itself to form the oxidized and more stable compounds DMDS and DMTS. In contrast, MTL was the dominant VSC produced in assays performed with CFEs (Fig. 2b). In this instance, some enzymes present in CFEs such as NADH oxidases could consume oxygen and partially prevent oxidation of MTL, making it more stable and more prevalent over longer periods (Fig. 2b). VSC production analyses were also carried out in 50 mM sodium citrate solution (pH 5.4 with 4.5% NaCl) to unveil whether MGL produced by the recombinant L. lactis pTnis-mgl strain was still active under cheese-ripening conditions. In this respect, when using CFEs of L. lactis pTnis-mgl, the levels of VSCs obtained were about 25% of the levels obtained under optimum conditions (data not shown). When whole cells of L. lactis pTnis-mgl were tested under these conditions, the levels of VSCs obtained remained 20% of those obtained at pH 8.0 (Fig. 3). A similar percentage was also observed when comparing the levels of VSC production obtained under the two study conditions with whole cells prepared from B. linens cultures (Fig. 3). Overall, these results clearly demonstrate that production of MGL in L. lactis can enhance the ability of these bacteria to produce VSCs, MGL being the enzyme still active under cheese-ripening conditions.

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FIG. 2. Production of the VSCs MTL, DMDS, and DMTS by coincubation of L-methionine with either purified recombinant His-tagged MGL or YtjE (a) or CFEs prepared from the recombinant L. lactis pTnis-mgl strain or the vector control L. lactis pTnis strain (b). Assays were carried out in 100 mM Tris HCl (pH 8.0) solution. Values are mg liter–1 mg–1 protein and are the means of three independent measurements. Standard errors are also indicated.

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FIG. 3. Production of the VSCs MTL, DMDS, and DMTS by coincubation of L-methionine with whole cells prepared from the recombinant L. lactis pTnis-mgl strain, the L. lactis pTnis strain carrying empty vector, or the B. linens strain adjusted to an OD600 of 20. Assays were carried out in 100 mM Tris HCl (pH 8.0) (a) or under cheese-ripening conditions (b) for 120 h. Values are mg liter–1 mg–1 protein and are the means of three independent measurements. Standard errors are also indicated.

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MGL has been reported as the key enzyme in the formation of VSCs by B. linens (2). This enzyme, however, has not been found in LAB, which have a limited capacity to produce VSCs (11, 15). Thus, the aim of this study was to bioengineer LAB to overproduce MGL from B. linens, in order to enhance their ability to produce MTL and other related VSCs known to be critical to cheese flavor development. Initial attempts to clone and express MGL in L. lactis failed as the native mgl gene was prone to rearrangement and/or deletions in this host. This may not be surprising as the G+C content of B. linens (G+C, 60 to 64%) is quite high (13) compared to that of L. lactis (G+C, 35.4 to 36.8%) (40) and these events can be more prevalent under these circumstances. G+C content variation is also the most important parameter differentiating codon bias between different organisms, and the frequencies of synonymous codon usage have been found to be species and even taxon specific (12). Furthermore, the expression of genes from organisms with a G+C content that is very different from that of the chosen expression system can often result in translational stalling and low yields of recombinant protein (6). Consequently, a second approach was used whereby a synthetic mgl gene was developed which had been codon adapted for optimal expression in L. lactis. This synthetic gene was successfully cloned and expressed as a heterologous protein (with or without His₆ tag) in L. lactis.

The purified MGL protein (with a His tag) was shown by SDS-PAGE to have a molecular mass of 47 kDa, in agreement with that predicted by its amino acid sequence (45.7 kDa). Furthermore, the protein was shown on native gels to have an apparent molecular mass of 170 kDa, comparable to that described previously with native MGL isolated from B. linens BL2, which is known to consist of four identical units of 43 kDa (16). In this respect, the recombinant enzyme is also similar to other MGL proteins that have been purified from Pseudomonas putida (31), Citrobacter freundii (29), and the primitive protozoa Entamoeba histolytica (37) and Trichomonas vaginalis (28).

The purified recombinant MGL protein (produced in L. lactis) was shown to degrade L-methionine, but at a 14-fold lower rate than that reported previously with purified native MGL from B. linens (16). We can only speculate, but the inclusion of the carboxyl-terminal histidine tag may have resulted in a reduction in the protein's biological activity. Furthermore, the recombinant His-tagged MGL protein was also able to degrade other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. This was unexpected as MGL from B. linens has been reported not to degrade L-cystathionine and L-cystine (16). One explanation for these differences is that previously the specificity of the native MGL enzyme was determined by measuring the -keto acid production instead of assaying for thiol group formation as described here. In terms of substrate specificity, the recombinant protein produced here does behave similarly to the purified MGL from T. vaginalis (28). Furthermore, apart from L-methionine and L-cysteine, other purified MGLs from Clostridium sporogenes (26) and Aeromonas sp. (20) have also been shown to degrade L-cysteine and L-cystathionine, respectively. When CFEs were tested, the L. lactis MGL strain was shown to demonstrate enzyme activities that were not only dramatically increased compared to the vector control strain but also similar to those obtained with CFEs from B. linens cultures.

The formation of VSCs due to the recombinant MGL activity toward L-methionine under optimum conditions but also under conditions that mimic cheese ripening was also confirmed. GC-MS analyses identified higher formation of MTL and its auto-oxidation products DMDS and DMTS when either purified MGL, whole cells, or CFEs from the overexpressing L. lactis strain were used. Compared to the YtjE C-S lyase, the recombinant L. lactis MGL yielded markedly larger amounts of VSCs, which is not surprising as the efficiency of L-methionine conversion by cystathionine β- and -lyases in L. lactis has been shown to be about 100-fold less than that of its preferred substrate, cystathionine (1, 11).

This is the first report of overexpression of mgl from B. linens, encoding MGL, in a food-grade microorganism such as L. lactis. In addition, this study shows activity of the recombinant MGL under cheese-ripening conditions, thus establishing an alternative basis for the control and/or enhancement of VSC synthesis during the ripening process. Other promising strategies aimed at enhancing cheese flavor, such as the overproduction of cystathionine lyases (22, 27) or the addition of B. linens cells to starter cultures (17), have not been developed further. In this work, the higher specificity toward L-methionine and other sulfur-containing compounds demonstrated by recombinant L. lactis strains producing MGL together with the considerable increase in VSC production point out that this strategy could represent a more efficient way to enhance cheese flavor development.

 
This work was supported by the Biotechnology and Biological Sciences Research Council (UK), the Research Project Intramural-200770I005 supported by CSIC-I3, and the Project AGL2006-12100 funded by the Spanish Ministerio de Ciencia e Innovación. M. C. Martínez-Cuesta was also awarded a Marina Bueno-Royal Society Fellowship.

 
* Corresponding author. Mailing address: Dept. of Dairy Science and Technology, Instituto del Frío, C/José Antonio Novais 10, Ciudad Universitaria, 28040 Madrid, Spain. Phone: 34915492300. Fax: 34915493627. E-mail: cmc{at}if.csic.es

Published ahead of print on 27 February 2009.

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

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1
1. Alting, A. C., W. J. M. Engels, S. van Schalkwijk, and F. A. Exterkate. 1995. Purification and characterization of cystathionine β-lyase from Lactococcus lactis subsp. cremoris B78 and its possible role in flavor development in cheese. Appl. Environ. Microbiol. 61:4037-4042.[Abstract]
2
2. Amarita, F., M. Yvon, M. Nardi, E. Chambellon, J. Delettre, and P. Bonnarme. 2004. Identification and functional analysis of the gene encoding methionine--lyase in Brevibacterium linens. Appl. Environ. Microbiol. 70:7348-7354.[Abstract/Free Full Text]
3
3. Amarita, F., T. Requena, G. Taborda, L. Amigo, and C. Pelaez. 2001. Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination. J. Appl. Microbiol. 90:971-978.[CrossRef][Medline]
4
4. Arfi, K., S. Landaud, and P. Bonnarme. 2006. Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens. Appl. Environ. Microbiol. 72:2155-2162.[Abstract/Free Full Text]
5
5. Aubel, D., J. E. Germond, C. Gilbert, and D. Atlan. 2002. Isolation of the patC gene encoding the cystathionine β-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis. Microbiology 148:2029-2036.[Abstract/Free Full Text]
6
6. Baca, A. M., and W. G. Hol. 2000. Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli. Int. J. Parasitol. 30:113-118.[CrossRef][Medline]
7
7. Bonnarme, P., C. Lapadatescu, M. Yvon, and H. E. Spinnler. 2001. L-Methionine degradation potentialities of cheese-ripening microorganisms. J. Dairy Res. 68:663-674.[CrossRef][Medline]
8
8. Bonnarme, P., F. Amarita, E. Chambellon, E. Semon, H. E. Spinnler, and M. Yvon. 2004. Methylthioacetaldehyde, a possible intermediate metabolite for the production of volatile sulphur compounds from L-methionine by Lactococcus lactis. FEMS Microbiol. Lett. 236:85-90.[Medline]
9
9. Bonnarme, P., L. Psoni, and H. E. Spinnler. 2000. Diversity of L-methionine catabolism pathways in cheese-ripening bacteria. Appl. Environ. Microbiol. 66:5514-5517.[Abstract/Free Full Text]
10
10. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
11
11. Bruinenberg, P. G., G. de Roo, and G. K. Y. Limsowtin. 1997. Purification and characterization of cystathionine -lyase from Lactococcus lactis subsp. cremoris SK11: possible role in flavor compound formation during cheese maturation. Appl. Environ. Microbiol. 63:561-566.[Abstract]
12
12. Chen, S. L., W. Lee, A. K. Hottes, L. Shapiro, and H. H. McAdams. 2004. Codon usage between genomes is constrained by genome-wide mutational processes. Proc. Natl. Acad. Sci. USA 101:3480-3485.[Abstract/Free Full Text]
13
13. Collins, M. D., D. Jones, R. M. Keddie, and P. H. A. Sneath. 1980. Reclassification of Chromobacterium iodinum (Davis) in a redefined genus Brevibacterium (Breed) as Brevibacterium iodinum nom. rev.; comb. nov. J. Gen. Microbiol. 120:1-10.[Abstract/Free Full Text]
14
14. de Ruyter, P. G. G. A., O. P. Kuipers, M. M. Beerthuyzen, I. van Alen-Boerrigter, and W. M. de Vos. 1996. Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis. J. Bacteriol. 178:3434-3439.[Abstract/Free Full Text]
15
15. Dias, B., and B. Weimer. 1998. Conversion of methionine to thiols by lactococci, lactobacilli, and brevibacteria. Appl. Environ. Microbiol. 64:3320-3326.[Abstract/Free Full Text]
16
16. Dias, B., and B. Weimer. 1998. Purification and characterization of L-methionine -lyase from Brevibacterium linens BL2. Appl. Environ. Microbiol. 64:3327-3331.[Abstract/Free Full Text]
17
17. Dias, B., and B. Weimer. 1999. Production of volatile sulphur compounds in Cheddar cheese slurries. Int. Dairy J. 9:605-611.[CrossRef]
18
18. Dobric, N., G. K. Limsowtin, A. J. Hillier, N. P. Dudman, and B. E. Davidson. 2000. Identification and characterization of a cystathionine β/-lyase from Lactococcus lactis ssp. cremoris MG1363. FEMS Microbiol. Lett. 182:249-254.[Medline]
19
19. Dodd, H. M., N. Horn, W. C. Chan, C. J. Giffard, B. W. Bycroft, G. C. K. Roberts, and M. J. Gasson. 1996. Molecular analysis of the regulation of nisin immunity. Microbiology 142:2385-2392.[Abstract/Free Full Text]
20
20. Esaki, N., and K. Soda. 1987. L-Methionine--lyase from Pseudomonas putida and Aeromonas. Methods Enzymol. 143:459-465.[Medline]
21
21. Ferchichi, M., D. Hemme, M. Nardi, and N. Pamboukgjian. 1985. Production of methanethiol from methionine by Brevibacterium linens CNRZ 918. J. Gen. Microbiol. 131:715-723.[Abstract/Free Full Text]
22
22. Fernández, M., W. van Doesburg, G. A. Rutten, J. D. Marugg, A. C. Alting, R. van Kranenburg, and O. P. Kuipers. 2000. Molecular and functional analyses of the metC gene of Lactococcus lactis, encoding cystathionine β-lyase. Appl. Environ. Microbiol. 66:42-48.[Abstract/Free Full Text]
23
23. Gao, S., E. S. Mooberry, and J. L. Steele. 1998. Use of ¹³C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci. Appl. Environ. Microbiol. 64:4670-4675.[Abstract/Free Full Text]
24
24. Gasson, M. J. 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-inducing curing. J. Bacteriol. 154:1-9.[Abstract/Free Full Text]
25
25. Kagkli, D. M., R. Tache, T. M. Cogan, C. Hill, S. Casaregola, and P. Bonnarme. 2006. Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem. Appl. Microbiol. Biotechnol. 73:434-442.[CrossRef][Medline]
26
26. Kreis, W., and C. Hession. 1973. Isolation and purification of L-methionine--deamino--mercaptomethane-lyase (L-methionase) from Clostridium sporogenes. Cancer Res. 33:1862-1865.[Abstract/Free Full Text]
27
27. Lee, W.-J., D. S. Banavara, J. E. Hughes, J. K. Christiansen, J. L. Steele, J. R. Broadbent, and S. A. Rankin. 2007. Role of cystathionine β-lyase in catabolism of amino acids to sulfur volatiles by genetic variants of Lactobacillus helveticus CNRZ 32. Appl. Environ. Microbiol. 73:3034-3039.[Abstract/Free Full Text]
28
28. Lockwood, B. C., and G. C. Coombs. 1991. Purification and characterization of methionine--lyase from Trichomonas vaginalis. Biochem. J. 279:675-682.[Medline]
29
29. Manukhov, I. V., D. V. Mamaeva, S. M. Rastorguev, N. G. Faleev, E. A. Morozova, T. V. Demidkina, and G. B. Zavilgesky. 2005. A gene encoding L-methionine -lyase is present in Enterobacteriaceae family genomes: identification and characterization of Citrobacter freundii L-methionine -lyase. J. Bacteriol. 187:3889-3893.[Abstract/Free Full Text]
30
30. Martínez-Cuesta, M. C., C. Peláez, J. Eagles, M. J. Gasson, T. Requena, and, S. Hanniffy. 2006. YtjE from Lactococcus lactis IL1403 is a C-S lyase with ,-elimination activity toward methionine. Appl. Environ. Microbiol. 72:4878-4884.[Abstract/Free Full Text]
31
31. Nakayama, T., N. Esaki, K. Sugie, T. T. Beresov, H. Tanaka, and K. Soda. 1984. Purification of bacterial L-methionine--lyase. Anal. Biochem. 138:421-424.[CrossRef][Medline]
32
32. Reuter, M. A., S. Hanniffy, and J. M. Wells. 2003. Expression and delivery of heterologous antigens using lactic acid bacteria. Methods Mol. Med. 87:101-104.[Medline]
33
33. Robinson, K., L. M. Chamberlain, K. M. Schofield, J. M. Wells, and R. W. Le Page. 1997. Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nat. Biotechnol. 15:653-657.[CrossRef][Medline]
34
34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
35
35. Seepersaud, R., S. B. Hanniffy, P. Mayne, P. Sizer, R. Le Page, and J. M. Wells. 2005. Characterization of a novel leucine-rich repeat protein antigen from group B streptococci that elicits protective immunity. Infect. Immun. 73:1671-1683.[Abstract/Free Full Text]
36
36. Soda, K. 1987. Microbial sulfur amino acids: an overview. Methods Enzymol. 143:453-459.[Medline]
37
37. Tokoro, M., T. Asai, S. Kobayashi, T. Takeuchi, and T. Nozaki. 2003. Identification and characterization of two isoenzymes of methionine--lyase from Entamoeba histolytica. J. Biol. Chem. 279:42717-42727.
38
38. Urbach, G. 1995. Contribution of lactic acid bacteria to flavour compound formation in dairy products. Int. Dairy J. 5:877-905.[CrossRef]
39
39. Uren, J. R. 1987. Cystathionine β-lyase from Escherichia coli. Methods Enzymol. 143:483-486.[Medline]
40
40. Wegmann, U., M. O'Connell-Motherway, A. Zomer, G. Buist, C. Shearman, C. Canchaya, M. Ventura, A. Goesmann, M. J. Gasson, O. P. Kuipers, D. van Sinderen, and J. Kok. 2007. Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363. J. Bacteriol. 189:3256-3270.[Abstract/Free Full Text]
41
41. Weimer, B., K. Seefeldt, and B. Dias. 1999. Sulfur metabolism in bacteria associated with cheese. Antonie van Leeuwenhoek 76:247-261.[CrossRef][Medline]
42
42. Wells, J. M., P. W. Wilson, and R. W. Le Page. 1993. Improved cloning vectors and transformation procedure for Lactococcus lactis. J. Appl. Bacteriol. 74:629-636.[Medline]
43
43. Yoshida, Y., Y. Nakano, A. Amano, M. Yoshimura, H. Fukamachi, T. Oho, and T. Koga. 2002. Lcd from Streptococcus anginosus encodes a C-S lyase with ,β-elimination activity that degrades L-cysteine. Microbiology 148:3961-3970.[Abstract/Free Full Text]
44
44. Zdych, E., R. Peist, J. Reidl, and W. Boos. 1995. MalY of Escherichia coli is an enzyme with the activity of a βC-S lyase (cystathionase). J. Bacteriol. 177:5035-5039.[Abstract/Free Full Text]

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Applied and Environmental Microbiology, April 2009, p. 2326-2332, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02417-08
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