Metabolic Engineering Strategies for High-Level α-Pinene Production in Saccharomyces cerevisiae

α-Pinene is a biologically active monoterpene compound derived from coniferous plants, performing multiple ecological and physiological functions. Moreover, its strained bicyclic skeleton makes it an exceptional renewable feedstock for high-density renewable fuels. However, the extraction of α-pinene from plants is a time-consuming and laborious process. Microbial cell factories offer a green, low-carbon, and tightly controllable route for producing α-pinene. Despite this, low biosynthetic efficiency limits the industrial production of α-pinene by microbial cells. In this study, we first demonstrated that SGE1, a transporter enhancing pinene synthesis, functions within the amino acid metabolic network, with its overexpression primarily promoting the synthesis and utilization of arginine and lysine. To enhance α-pinene synthesis, the Pt30^S122N mutant was obtained, resulting in a 34.4% increase in α-pinene yield. Differential expression analysis between isopropyl myristate (IPM)-supplemented and IPM-free engineered strains identified AGP1, an amino acid transporter that enhances production in pinene engineered strains, with α-pinene titers increasing by up to 14% upon its overexpression. Ultimately, diploid fusion further boosted production by 88.5% over the haploid counterpart in a 5-L bioreactor, achieving the highest yeast titer reported to date of 3.9 g/L. This study establishes a platform for the microbial industrial production of α-pinene.