Regulatory Networks Governing Methionine Catabolism into Volatile Organic Sulfur-Containing Compounds in Clonostachys rosea.

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.

Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1.

Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering.

Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol.

Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging.