Mushroom Derived STS-HMGS Fusion Enzyme Enhances Sesquiterpene Production in Fungal Hosts

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is a key enzyme in the mevalonate (MVA) pathway that catalyzes the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) from acetoacetyl-CoA and acetyl-CoA. Recently, a novel class of HMGS-sesquiterpene synthase (STS) fusion enzymes has been identified. In this study, we discovered a natural fusion enzyme, GihirA, in <i>Gloeostereum incarnatum</i>, which contains both STS and HMGS domains and synthesizes the sesquiterpenoid hirsutene. Our investigation revealed that the HMGS domain significantly enhances the cyclization activity of the STS domain, resulting in an 8.87-fold increase in sesquiterpene production with a final yield of 121.3 mg/kg, highlighting HMGS's critical role in catalytic efficiency. Additionally, domain-swapping experiments were performed by replacing the HMGS domain of <i>G. incarnatum</i> with the native HMGS domain from <i>Flammulina velutipes</i> sesquiterpene synthase Fla2. The results demonstrated that Fla2 fused with its cognate HMGS domain exhibited a significant yield enhancement from 11.5 to 54.5 mg/kg, underscoring the importance of metabolic compatibility in enzyme performance. This study not only reveals the unique advantages of natural fusion enzymes in sesquiterpene biosynthesis but also provides an important theoretical foundation for enhancing sesquiterpene production through the optimization of enzyme fusion strategies and metabolic pathway design. These findings offer a rational strategy for engineering high-efficiency terpenoid biosynthesis.