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Transcriptional analysis of L-methionine catabolism in the cheese-ripening yeast Yarrowia lipolytica in relation to volatile sulfur compound biosynthesis.

ABSTRACT: 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 YlARO8 (YALI0E20977g), YlBAT1 (YALI0D01265g), and YlBAT2 (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 YlPDB1 (YALI0E27005g) and YlPDC6 (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 YlARO8, YlBAT1, and YlBAT2 genes could play a key role in this process.

SUBMITTER: Cholet O

PROVIDER: S-EPMC2423039 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Transcriptional analysis of L-methionine catabolism in the cheese-ripening yeast Yarrowia lipolytica in relation to volatile sulfur compound biosynthesis.

Cholet Orianne O Hénaut Alain A Hébert Agnès A Bonnarme Pascal P

Applied and environmental microbiology 20080404 11

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 p ...[more]

PMID: 18390675

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Project description:Adaptation to environmental perturbations requires living systems to coordinately regulate signaling pathways, gene expression, and metabolism. To better understand the mechanisms underlying adaptation, the regulatory nodes within networks must be elucidated. Here, ARO8-2 (which encodes an aminotransferase), PDC (which encodes a decarboxylase), and STR3 (which encodes a demethiolase) were identified as key genes involved in the catabolism of methionine in the mycoparasitic fungus Clonostachys rosea, isolated from Tuber melanosporum ascocarps. Exogenous Met induced the transcription of ARO8-2 and PDC but repressed the transcription of STR3, which is controlled by the putative MSN2 and GLN3 binding sites responding to nitrogen catabolite repression. Met and its structural derivatives function as glutamine synthetase inhibitors, resulting in the downregulation of STR3 expression. The putative GLN3 binding site was necessary for STR3 downregulation. In Saccharomyces cerevisiae, Met and its structural derivatives also triggered downregulation of demethiolase gene expression. Altogether, the results indicated that exogenous Met triggered nitrogen catabolite repression, which stimulated the Ehrlich pathway and negatively regulated the demethiolation pathway via the methionine sulfoximine-responsive regulatory pathway. This finding revealed the regulatory nodes within the networks controlling the catabolism of Met into volatile organic sulfur-containing compounds, thereby enhancing our understanding of adaptation.IMPORTANCE Methionine shuttles organic nitrogen and plays a central role in nitrogen metabolism. Exogenous Met strongly induces the expression of ARO8-2 and PDC, represses the expression of STR3, and generates volatile organic sulfur-containing compounds via the Ehrlich and demethiolation pathways. In this study, we used genetic, bioinformatic, and metabolite-based analyses to confirm that transcriptional control of the aminotransferase gene ARO8-2, the decarboxylase gene PDC, and the demethiolase gene STR3 modulates Met catabolism into volatile organic sulfur-containing compounds. Importantly, we found that, in addition to the Ehrlich pathway, the demethiolation pathway was regulated by a nitrogen catabolite repression-sensitive regulatory pathway that controlled the transcription of genes required to catabolize poor nitrogen sources. This work significantly advances our understanding of nitrogen catabolite repression-sensitive transcriptional regulation of sulfur-containing amino acid catabolism and provides a basis for engineering Met catabolism pathways for the production of fuel and valuable flavor alcohols.

Project description:Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push-pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.

Dataset Information

Transcriptional analysis of L-methionine catabolism in the cheese-ripening yeast Yarrowia lipolytica in relation to volatile sulfur compound biosynthesis.

Publications

Transcriptional analysis of L-methionine catabolism in the cheese-ripening yeast Yarrowia lipolytica in relation to volatile sulfur compound biosynthesis.

Similar Datasets

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