Methylthioacetaldehyde, a possible intermediate metabolite for the production of volatile sulphur compounds from -methionine by Lactococcus lactis

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 sulfurcontaining 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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confidence: 99%

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

Hanniffy

Philo

Peláez

et al. 2009

“…In LAB, L-methionine degradation could be initiated by a transamination step that leads to 4-methylthio-2-oxobutyric acid (MOBA), which is subsequently converted to MTL through a chemical process (6,10). In Lactococcus lactis, two aminotransferases (17), one aromatic aminotransferase and one branched-chain aminotransferase, were shown to be involved in the synthesis of volatile aroma compounds but can catalyze the transamination of L-methionine to MOBA only to a limited extent.…”

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confidence: 99%

Evidence for Distinct l -Methionine Catabolic Pathways in the Yeast Geotrichum candidum and the Bacterium Brevibacterium linens

Arfi

Landaud

Bonnarme

2006

Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and ␣-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G. candidum could also be used as precursors for VSC production by B. linens.Traditional fermented products result from the action of a set of microorganisms that give these products their diversity, their uniqueness, and their quality. In this context, sulfur compounds play a key role (18). Owing to their low detection thresholds and their strong reactivity, volatile sulfur compounds (VSCs) significantly influence the quality and unique flavor characteristics of many foodstuffs, such as cheese, wine, and beer (15,16,20). VSCs essentially arise from degradation of the sulfur-carbon bond of the amino acid precursor L-methionine, giving rise to methanethiol (MTL), a common precursor of VSCs (5).A large number of microorganisms, including yeasts and bacteria, are employed in traditional food processing, such as cheese ripening and wine and beer making, and are often used in association with each other. In soft cheeses, yeasts (e.g., Geotrichum candidum) develop in the early stages of cheese ripening and participate in the deacidification of the curd and, to a lesser extent, in aromatization (2). In contrast, acidsensitive surface bacteria (e.g., Brevibacterium linens) develop much later than yeasts during ripening, once the pH is sufficiently raised by the deacidifying yeast (3). B. linens is known to produce consistent amounts of VSCs (8, 9). L-Methionine ␥-lyase (MGL) catalyzes the one-step degradation of L-methionine to MTL, ␣-ketobutyrate (␣-KB), and ammonium, and the gene encoding this enzyme in B. linens has been identified recently (1). In contrast, lactic acid bacteria (LAB) poorly convert L-methionine to VSCs (9) because they possess Met aminotransferase but no MGL activity. It therefore seem...

“…In contrast, in LAB the reaction is catalyzed by a cystathionine ␤-lyase (CBL) and a cystathionine ␥-lyase (CGL) which are only slightly active towards L-methionine (1, 10, 18). In LAB, another pathway for L-methionine conversion to VSCs also exists but produces limited amounts of MTL (7,35).Coryneform bacteria are generally found on the surface of smear cheeses and give the typical sulfur notes to cheeses such as Limburger, Tilsiter, Livarot, Epoisses, and Munster. To date, B. linens is the only food-grade bacterium from which MGL has been purified and characterized (16,26,31,38,39), but neither its protein sequence nor its gene sequence are presently available.…”

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confidence: 99%

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confidence: 99%

Identification and Functional Analysis of the Gene Encoding Methionine-γ-Lyase in Brevibacterium linens

Amárita

Yvon²,

Nardi³

et al. 2004

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-Methionine-␥-lyase (MGL) can convert Lmethionine to methanethiol (MTL), ␣-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiolproducing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via ␥-elimination is a key step in the formation of VSCs in B. linens.Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to the typical aromas of different cheeses (12,14,33). VSCs arise primarily from the degradation of L-methionine to methanethiol (MTL) by the cheese microflora. This thiol is a common precursor for a variety of other sulfur-bearing compounds including the auto-oxidation products (11), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters, primarily arising from chemical reaction of MTL with acyl coenzyme A (acyl-CoA) (22). Numerous studies have therefore been done to control and/or diversify VSC synthesis during the ripening process by the use of properly selected microorganisms (4, 6, 15, 43). Many cheese microorganisms are capable of producing VSCs from L-methionine. Some of them, such as brevibacteria, especially Brevibacterium linens (17), are known to be very good VSC producers while others, such as lactic acid bacteria (LAB), can produce only limited amounts of VSCs (14).The most direct route for MTL biosynthesis, is the L-methionine ␥-elimination that directly produces MTL, ␣-ketobutyrate, and ammonia from L-methionine. This L-methionine ␥-elimination activity is quite high in B. linens and corynebacteria (17) and is also suspected in several other cheese surface bacteria, such as Micrococcus luteus, Arthrobacter sp., and Staphylococcus equorum (8). In contrast, such activity is quite low in LAB (14). In B. linens, the methionine ␥-elimination is catalyzed by a L-methionine-␥-lyase (MGL), a pyridoxal phosphate (PLP)-dependent enzyme for which L-methionine is the best substrate (16). In contrast, in LAB the reaction is catalyzed by a cystathionine ␤-lyase (CBL) and a cystathionine ␥-lyase (CGL) which are only slightly active towards L-methionine (1, 10, 18). In LAB, another pathway for L-methionine conversion to VSCs also exists but produces limited amounts of MTL (7,35).Coryneform bacteria are generally found on the surface of smear cheeses and give the typical sulfur notes to cheeses such as Limburger, Tilsiter, Livarot, Epoisses, and Munster. To date, B. linens is the only food-grade bacterium from which MGL has been purified and characterized (16,26,31,38,39), but neither its protein sequence nor its gene...