Metabolic modulation of TCA cycle by S-nitrosylation in Monascus spp.

<i>Monascus</i> spp. produces several beneficial secondary metabolites (SMs), such as Monacolin K and Monascus pigments, which have wide applications in the pharmaceutical and food industries. Various factors, including gene expression, regulate the metabolic processes of <i>Monascus</i> spp. Furthermore, <i>S</i>-nitrosylation also modulates the activity of its metabolic enzymes. However, the specific regulatory role and mechanisms of S-nitrosylation in the metabolic pathways of <i>Monascus</i> spp. remain unexplored. In this study, quantitative <i>S</i>-nitrosoproteomics of <i>M. purpureus</i> identified 352 modified proteins, representing 12% of the total protein content in <i>M. purpureus</i> HJ11. <i>S</i>-nitrosylated proteins were predominantly distributed in glycolysis, tricarboxylic acid (TCA) cycle, amino acid metabolism and fatty acid metabolism. Notably, 87% of enzymes in the TCA cycle were <i>S</i>-nitrosylated. Further correlation analyses and in vitro enzymatic experiments suggest that protein <i>S</i>-nitrosylation inhibits the TCA cycle, thereby reducing the production of Monacolin K and <i>Monascus</i> pigments. To our knowledge, these findings demonstrate for the first time that <i>S</i>-nitrosylation serves as a key metabolic regulator in <i>Monascus</i> spp. This study elucidated how nitrogen-source-modulated protein <i>S</i>-nitrosylation inhibits the TCA cycle in <i>Monascus</i> spp., exerting differential effects on cell growth and the metabolic profile, including the biosynthesis of SMs, amino acids, and other key products. Our study also provides novel insights into enhancing high-value-added metabolite production in <i>Monascus</i> spp.