Pascal Bonnarme

Despite their importance as potent odors that contribute to the aroma of numerous cheeses, S-methyl thioesters formation pathways have not been fully established yet. In a first part of our work, we demonstrated that Brevibacterium antiquum and Brevibacterium aurantiacum could produce S-methyl thioesters using short-chain fatty acids or branched-chain amino acids as precursors. Then, we focused our work on l-leucine catabolism using liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry analyses coupled with tracing experiments. For the first time, several acyl–CoAs intermediates of the l-leucine to thioesters conversion pathway were identified. S-methyl thioisovalerate was produced from l-leucine, indicating that this amino acid was initially transaminated. Quite interestingly, data also showed that other S-methyl thioesters, e.g., S-methyl thioacetate or S-methyl thioisobutyrate, were produced from l-leucine. Enzymatic and tracing experiments allowed for postulating catabolic pathways leading to S-methyl thioesters biosynthesis.

In this study, we combined metabolic reconstruction, growth assays, and metabolome and transcriptome analyses to obtain a global view of the sulfur metabolic network and of the response to sulfur availability inBrevibacterium aurantiacum. In agreement with the growth ofB. aurantiacumin the presence of sulfate and cystine, the metabolic reconstruction showed the presence of a sulfate assimilation pathway, thiolation pathways that produce cysteine (cysEandcysK) or homocysteine (metXandmetY) from sulfide, at least one gene of the transsulfuration pathway (aecD), and genes encoding three MetE-type methionine synthases. We also compared the expression profiles ofB. aurantiacumATCC 9175 during sulfur starvation or in the presence of sulfate. Under sulfur starvation, 690 genes, including 21 genes involved in sulfur metabolism and 29 genes encoding amino acids and peptide transporters, were differentially expressed. We also investigated changes in pools of sulfur-containing metabolites and in expression profiles after growth in the presence of sulfate, cystine, or methionine plus cystine. The expression of genes involved in sulfate assimilation and cysteine synthesis was repressed in the presence of cystine, whereas the expression ofmetX,metY,metE1,metE2, andBL613, encoding a probable cystathionine-γ-synthase, decreased in the presence of methionine. We identified three ABC transporters: two operons encoding transporters were transcribed more strongly during cysteine limitation, and one was transcribed more strongly during methionine depletion. Finally, the expression of genes encoding a methionine γ-lyase (BL929) and a methionine transporter (metPS) was induced in the presence of methionine in conjunction with a significant increase in volatile sulfur compound production.

The interactions that may occur between microorganisms in different ecosystems have not been adequately studied yet. We investigated yeast-bacterium interactions in a synthetic medium using different culture associations involving the yeast Yarrowia lipolytica 1E07 and two bacteria, Staphylococcus xylosus C2a and Lactococcus lactis LD61. The growth and biochemical characteristics of each microorganism in the different culture associations were studied. The expression of genes related to glucose, lactate, and amino acid catabolism was analyzed by reverse transcription followed by quantitative PCR. Our results show that the growth of Y. lipolytica 1E07 is dramatically reduced by the presence of S. xylosus C2a. As a result of a low amino acid concentration in the medium, the expression of Y. lipolytica genes involved in amino acid catabolism was downregulated in the presence of S. xylosus C2a, even when L. lactis was present in the culture. Furthermore, the production of lactate by both bacteria had an impact on the lactate dehydrogenase gene expression of the yeast, which increased up to 30-fold in the three-species culture compared to the Y. lipolytica 1E07 pure culture. S. xylosus C2a growth dramatically decreased in the presence of Y. lipolytica 1E07. The growth of lactic acid bacteria was not affected by the presence of S. xylosus C2a or Y. lipolytica 1E07, although the study of gene expression showed significant variations.

The yeast Yarrowia lipolytica has to develop dynamic metabolic adaptation mechanisms to survive within the cheese habitat. The availability of amino acids (AAs) is of major importance for microbial development and/or aroma production during cheese ripening. Using 2‐D protein gel electrophoresis, we analyzed the adaptation mechanisms of Y. lipolytica for AAs limitation or supplementation in a batch culture containing lactate as a carbon source. Proteome analyses allow the identification of 34 differentially expressed proteins between the culture conditions. These analyses demonstrated that prior to the AAs addition, mainly proteins involved in the oxidative stress of the yeast were induced. Following the AAs addition, yeast cells reorganize their metabolism toward AAs catabolism and also generate a higher induction of proteins related to carbon metabolism and proteins biosynthesis. Using real‐time reverse transcription PCR, we re‐evaluated the expression of genes encoding proteins involved in these processes. The expression levels of the genes were in accordance with the proteomic results, with the up‐regulation of genes encoding a branched‐chain amino transferase BAT2, a pyruvate decarboxylase PDC6 and an Hsp70 protein SSZ1 involved in protein biosynthesis. A volatile compound analysis was also performed, and increased production of dimethyldisulfide from methionine and 3‐methyl‐butanal from leucine was observed in media supplemented with AAs.

The consumption of lactate and amino acids is very important for microbial development and/or aroma production during cheese ripening. A strain of Yarrowia lipolytica isolated from cheese was grown in a liquid medium containing lactate in the presence of a low (0.1×) or high (2×) concentration of amino acids. Our results show that there was a dramatic increase in the growth of Y. lipolytica in the medium containing a high amino acid concentration, but there was limited lactate consumption. Conversely, lactate was efficiently consumed in the medium containing a low concentration of amino acids after amino acid depletion was complete. These data suggest that the amino acids are used by Y. lipolytica as a main energy source, whereas lactate is consumed following amino acid depletion. Amino acid degradation was accompanied by ammonia production corresponding to a dramatic increase in the pH. The effect of adding amino acids to a Y. lipolytica culture grown on lactate was also investigated. Real-time quantitative PCR analyses were performed with specific primers for five genes involved in amino acid transport and catabolism, including an amino acid transporter gene ( GAP1 ) and four aminotransferase genes ( ARO8 , ARO9 , BAT1 , and BAT2 ). The expression of three genes involved in lactate transport and catabolism was also studied. These genes included a lactate transporter gene ( JEN1 ) and two lactate dehydrogenase genes ( CYB2 - 1 and CYB2 - 2 ). Our data showed that GAP1 , BAT2 , BAT1 , and ARO8 were maximally expressed after 15 to 30 min following addition of amino acids ( BAT2 was the most highly expressed gene), while the maximum expression of JEN1 , CYB2 - 1 , and CYB2 - 2 was delayed (≥60 min).

Yarrowia lipolytica is one of the yeasts most frequently isolated from the surface of ripened cheeses. In previous work, it has been shown that this yeast is able to convert l -methionine into various volatile sulfur compounds (VSCs) that may contribute to the typical flavors of several cheeses. In the present study, we show that Y. lipolytica does not assimilate lactate in the presence of l -methionine in a cheeselike medium. Nineteen presumptive genes associated with l -methionine catabolism or pyruvate metabolism in Y. lipolytica were transcriptionally studied in relation to l -methionine degradation. The expression levels of the Yl ARO8 (YALI0E20977g), Yl BAT1 (YALI0D01265g), and Yl BAT2 (YALI0F19910g) genes (confirmed by real-time PCR experiments) were found to be strongly up-regulated by l -methionine, and a greater variety and larger amounts of VSCs, such as methanethiol and its autooxidation products (dimethyl disulfide and dimethyl trisulfide), were released in the medium when Y. lipolytica was grown in the presence of a high concentration of l -methionine. In contrast, other genes related to pyruvate metabolism were found to be down-regulated in the presence of l -methionine; two exceptions were the Yl PDB1 (YALI0E27005g) and Yl PDC6 (YALI0D06930g) genes, which encode a pyruvate dehydrogenase and a pyruvate decarboxylase, respectively. Both transcriptional and biochemical results corroborate the view that transamination is the first step of the enzymatic conversion of l -methionine to VSCs in Y. lipolytica and that the Yl ARO8 , Yl BAT1 , and Yl BAT2 genes could play a key role in this process.

The formation of volatile sulfur compounds (VSC) in fermented food is a subject of interest. Such compounds are essential for the aroma of many food products like cheeses or fermented beverages, in which they can play an attractive or a repulsive role, depending on their identity and their concentration. VSC essentially arise from common sulfur-bearing precursors, methionine being the most commonly found. In the first section of this paper, the main VSC found in cheese, wine, and beer are reviewed. It is shown that a wide variety of VSC has been evidenced in these food products. Because of their low odor threshold and flavor notes, these compounds impart essential sensorial properties to the final product. In the second section of this review, the main (bio)chemical pathways leading to VSC synthesis are presented. Attention is focused on the microbial/enzymatic phenomena—which initiate sulfur bearing precursors degradation—leading to VSC production. Although chemical reactions could also play an important role in this process, this aspect is not fully developed in our review. The main catabolic pathways leading to VSC from the precursor methionine are presented.

DNA microarrays of 86 genes from the yeastsDebaryomyces hansenii,Kluyveromyces marxianus, andYarrowia lipolyticawere developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of theLACgenes ofK. marxianuswas observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially inD. hanseniiandK. marxianus, which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved inl-methionine catabolism was also used on the array.Y. lipolytica, which efficiently assimilatesl-methionine, also exhibited a high expression of theSaccharomyces cerevisiaeorthologsBAT2andARO8, which are involved in thel-methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures.

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 γ-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 α-ketoacid dehydrogenase and one gene encoding an acetolactate synthase were also up-regulated by l-methionine, and are probably involved in the catabolism of α-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.