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

Abstract

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...

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“…4-methylthio-2-oxobutyric acid (KMBA). It has been characterized in a variety of animals (22), in yeasts (23,24), and in Lactococcus lactis (25). [ 13 C]-NMR studies performed in yeast cells indicated that KMBA was mainly decarboxylated then reduced to produce methionol or, in a lower extent, cleaved by a demethiolase activity to produce CH 3 SH and ␣KB.…”

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“…To compensate the secondary loss of MGL, these bacteria might utilize an alternative pathway, as shown in yeast G. candidum. In this pathway, methionine degradation is initiated by transamidation, which forms 4-methylthio-2-oxobutyrate, followed by a cleavage to yield 2-oxobutyrate and methanethiol by chemical process (44). The comparative metabolomics and fluxomics between the bacteria possessing or lacking MGL should help our understanding its biological importance of MGLs.…”

“…Methanethiol is converted to dimethyl disulfide, dimethyl trisulfide, and thioesters, and 2-oxobutyrate is condensed with active acetaldehyde derived from pyruvate, and metabolized to 2,3-pentanedione (acetyl propionyl) (44). This condensation reaction is catalyzed by acetolactate synthase (44,45).…”

“…In an acidophilic iron-oxidizing euryarchaeon Ferroplasma acidarmanus, methanethiol has been demonstrated, while its draft genome apparently lacks a gene encoding MGL or similar enzymes (The UCSC Archaeal Genome Browser at http://archaea.ucsc.edu/). Thus, it is feasible that in F. acidarmanus, methionine is degraded in two step reactions consisting an amino transfer, which yields 4-methylthio-2-oxobutyric acid, and chemical decompose into 2-oxobutyrate, as proposed for methionine catabolic pathway in yeast Geotrichum candidum (44).…”

“…In the cheese ripening bacterium B. linens, methanethiol and 2-oxobutyrate are produced by MGL (44). Methanethiol is converted to dimethyl disulfide, dimethyl trisulfide, and thioesters, and 2-oxobutyrate is condensed with active acetaldehyde derived from pyruvate, and metabolized to 2,3-pentanedione (acetyl propionyl) (44).…”