Enhancing Monacolin K and GABA Biosynthesis in Monascus pilosus via GAD Overexpression: Multi-Omics Elucidation of Regulatory Mechanisms

<i>Monascus</i> produces various bioactive compounds, including monacolin K (MK), γ-aminobutyric acid (GABA), and <i>Monascus</i> pigments (MPs). Studies have shown that overexpressing genes within the MK biosynthetic cluster significantly enhances MK production. Additionally, MK synthesis in <i>Monascus</i> is regulated by other genes. Based on previous transcriptomic analyses conducted in our laboratory, a significant positive correlation was identified between the expression level of the <i>GAD</i> gene and MK production in <i>M. pilosus</i>. In this study, the <i>GAD</i> gene from <i>M. pilosus</i> was selected for overexpression, and a series of engineered <i>M. pilosus</i> strains were constructed. Among the 20 PCR-positive transformants obtained, 13 strains exhibited MK production increases of 12.84-52.50% compared to the parental strain, while 17 strains showed GABA production increases of 17.47-134.14%. To elucidate the molecular mechanisms underlying the enhanced production of MK and GABA, multi-omics analyses were performed. The results indicated that <i>GAD</i> overexpression likely promotes MK and GABA synthesis in <i>M. pilosus</i> by regulating key genes (e.g., <i>HPD</i>, <i>HGD</i>, and <i>FAH</i>) and metabolites (e.g., α-D-ribose-1-phosphate, β-alanine) involved in pathways such as tyrosine metabolism, phenylalanine metabolism, the pentose phosphate pathway, propanoate metabolism, and β-alanine metabolism. These findings provide theoretical insights into the regulatory mechanisms of MK and GABA biosynthesis in <i>Monascus</i> and suggest potential strategies for enhancing their production.