Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens

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 alpha-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.     

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

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-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-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 thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

Diversity of L-methionine catabolism pathways in cheese-ripening bacteria

Enzymatic activities that could be involved in methanethiol generation in five cheese-ripening bacteria were assayed, and the major sulfur compounds produced were identified. L-Methionine and alpha-keto-gamma-methyl-thio-butyric acid demethiolating activities were detected in whole cells and cell extracts (CFEs) of all the bacteria tested. No L-methionine deaminase activity could be detected in any of the ripening bacteria and L-methionine aminotransferase was detected in CFEs of Brevibacterium linens, Micrococcus luteus, and Corynebacterium glutamicum. The results suggest that several pathways for L-methionine catabolism probably coexist in these ripening bacteria.     

Involvement of a branched-chain aminotransferase in production of volatile sulfur compounds in Yarrowia lipolytica

The enzymatic degradation of L-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast Yarrowia lipolytica, the degradation of L-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (YlBCA1) of Y. lipolytica was amplified, and the L-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that L-methionine degradation via transamination is involved in formation of VSCs in Y. lipolytica.     

L-methionine degradation pathway in Kluyveromyces lactis: identification and functional analysis of the genes encoding L-methionine aminotransferase

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of L-methionine. L-methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, KlARO8.1 and KlARO8.2, were found to be responsible for L-methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.     

The physiology of L-methionine catabolism to the secondary metabolite ethylene by Escherichia coli

Catabolism of L-methionine by Escherichia coli strain B SPAO led to the formation of ethylene as a secondary metabolite (ethylenogenesis). Methionine was initially deaminated by a transamination reaction to the 2-oxo acid 2-oxo-4-methylthiobutyric acid (KMBA) which was then converted to ethylene. The utilization of L-methionine as an additional nitrogen source was investigated by examining ethylene synthesis under different nitrogen supply conditions. Ethylene formation in batch culture was unaffected by the concentration of the precursor L-methionine in the medium although increasing concentrations of NH4Cl resulted in progressively less ethylene formation. Cultures grown without L-methionine did not produce ethylene but were able to synthesize ethylene when L-methionine or KMBA was provided. Addition of L-tyrosine to batch cultures reduced the yield of ethylene after 42 h by 54%. Under these conditions the maximum transient level of KMBA was reduced by 32% and occurred later compared to when L-methionine was the only amino acid supplement. Continuous cultures grown under ammonia limitation produced both ethylene and KMBA. In contrast, when glucose was limiting, neither of these metabolites were produced. Cells harvested from continuous cultures grown under glucose or ammonia limitation were able to synthesize ethylene from either L-methionine or KMBA although their capacity for ethylene synthesis (ethylenogenic capacity) was optimal under ammonia limitation (C:N ratio = 20).     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Transcriptional analysis of L-methionine catabolism in Brevibacterium linens ATCC9175

The expression of genes possibly involved in L-methionine and lactate catabolic pathways were performed in Brevibacterium linens (ATCC9175) in the presence or absence of added L-methionine. The expression of 27 genes of 39 selected genes differed significantly in L-methionine-enriched cultures. The expression of the gene encoding L-methionine gamma-lyase (MGL) is high in L-methionine-enriched cultures and is accompanied by a dramatic increase in volatile sulfur compounds (VSC) biosynthesis. Several genes encoding alpha-ketoacid dehydrogenase and one gene encoding an acetolactate synthase were also up-regulated by L-methionine, and are probably involved in the catabolism of alpha-ketobutyrate, the primary degradation product of L-methionine to methanethiol. Gene expression profiles together with biochemical data were used to propose catabolic pathways for L-methionine in B. linens and their possible regulation by L-methionine.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Development and validation of a plate technique for screening of microorganisms that produce volatile sulfur compounds

Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow-orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.     

Dual influence of the carbon source and <Emphasis Type="SmallCaps">l</Emphasis>-methionine on the synthesis of sulphur compounds in the cheese-ripening yeast<Emphasis Type="Italic"> Geotrichum candidum</Emphasis>

The effect of the carbon source and l-methionine on the ability of Geotrichum candidum to produce volatile sulphur compounds (VSC) was studied. This yeast was cultivated in a synthetic medium supplemented with various carbon sources and l-methionine at different concentrations. Both glycerol and glucose significantly increased VSC production by G. candidum. Unlike the effect on the l-methionine- and 4-methylthio-2-oxobutyric acid-demethiolating activities, the supply of a carbon source had a dramatic effect on the activity of aminotransferase, a key enzyme in l-methionine catabolism. An increase in the initial concentration of l-methionine resulted in a rise in the production of sulphur compounds (VSC, 4-methylthio-2-oxobutyric acid) but had limited effect on l-methionine-catabolising enzyme activities. Evidence for the existence of a dual effect of the carbon source and l-methionine on VSC biosynthesis was obtained in this study.     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Effect of substrate-dependent microbialy produced ethylene on plant growth

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C2H4) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC2) for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic "triple" response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbialy produced ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC2 (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC2 in descending order. Results further revealed that ethylene accumulation in soil released from these substrates created a classic "triple" response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic "triple" response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC2, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived [C2H4] and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction.     

Improvement of the cryopreservation of the fungal starter Geotrichum candidum by artificial nucleation and temperature downshift control

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.     

Methionine transamination—metabolic function and subcellular compartmentation

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.     

The effect of indole-3-Acetic acid on ethylene formation in wheat seedlings

Isoperoxidase B 1 isolated from winter wheat (Triticum aestivum L., cv. Jubilar) seedlings was shown to catalyze ethylene formation from α-keto, γ-methylmercaptobutyric acid (KMBA). In the presence of Mn²⁺, indole-3-acetic acid (IAA), andp-coumaric acid, the kinetics by isoperoxidase B 1 catalyzed conversion of KMBA into ethylene and other products was similar to that of IAA oxidation. The reaction rate was therefore controlled by IAA through its electrondonating properties. Exogenous IAA induced ethylene formation in the segments of etiolated wheat coleoptiles. IAA-induced ethylene production was enhanced by L-methionine and mitomycin C. Aminoethoxy-analogue of rhizobitoxine, ferulic acid, sodium benzoate, cycloheximide and actinomyoin D exhibited significant inhibitory effects. These data indicate that the overall reaction mechanism in coleoptile segments involves RNA and protein synthesis. The site of IAA action is not specific; 2,4-dichlorophenoxyacetic, α-naphthylacetic and indole-3-butyric acids, respectively, possessed comparable inductive effect as IAA. Indole-3-propionic acid, indole, L-tryptophan and glucobrassicin had only low inductive efficiency, and moreover indole and L-tryptophan slowed down IAA-induced ethylene formation.     

The occurrence of 4-methylthio-2-hydroxybutyrate in human urine

A method for determination of 4-methylthio-2-hydroxybutyrate and 4-methylthio-2-oxobutyrate in human urine has been devised, based on metoxime formation of the keto acid and a clean-up procedure using a strong anion-exchange resin AG 2 X 8 and ethyl acetate extraction. After alkylation, the compounds were quantified by GC, using a flame photometric sulfur-selective detector. A normal urinary excretion of 0.14 to 0.25 mmol/mol creatinine and 0.07 to 0.22 mmol/mol creatinine of the alpha-hydroxy and alpha-keto acid, respectively, was found, whereas a markedly elevated excretion of the hydroxy acid was noted in subjects with hypermethioninemia. The enzymatic reduction of 4-methylthio-2-oxobutyric acid by lactate dehydrogenase: NAD+ oxidoreductase (EC 1.1.1.17) was also studied. The Km and Kequil values for 4-methylthio-2-oxobutyrate were 1.41 mM and 0.92 X 10(8) M-1. The Vmax value of the enzyme at infinite concentrations of the two substrates was 7.2 mumol/s/mumol enzyme, which indicates low affinity and reduced catalytic activity compared to other known substrates of lactate dehydrogenase. The reaction product 4-methylthio-2-hydroxybutyrate was not inhibitory on the reaction. The M4 isoenzyme of lactate dehydrogenase (rabbit and pig muscle) possessed approximately 20% of the activity of the H4 isoenzyme (pig heart) for the substrate.     

Resin straw as an alternative system to securely store frozen microorganisms

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.