Global Metabolic Rewiring Induced by HAC1 Overexpression Facilitates Efficient Linalool Synthesis in Saccharomyces cerevisiae

The present study employs global transcriptional engineering (GTE) to optimize metabolic flux toward linalool synthesis by screening seven key transcription factors in Saccharomyces cerevisiae. Overexpression of HAC1 in the strain SA44, the master regulator of the unfolded protein response (UPR), increased 2.9-fold in linalool titers compared to the control strain (SA49). Genome-wide transcriptomic analysis revealed that Hac1p-mediated activation induces a redirection of carbon flux into the mevalonate (MVA) pathway, the glyoxylate cycle, and gluconeogenesis, alongside the substantial repression of master sterol pathway activator (UPC2) and the hexose transporters. Multi-layer network analysis revealed GCN4- centered upregulation of aminoacyl-tRNA synthetase genes (aaRS) in the HAC1- overexpressing background, associated with putative HAC1-linked transcriptional repression of ERG9. Functional validation of the Hac1p regulatory networks via co-expression of HAC1 with the succinate-fumarate transporter (SFC1) yielded a synergistic improvement in linalool titer. Furthermore, the co-expression of MAF1, a negative regulator of tRNA biogenesis, and HAC1 has been shown to reduce linalool production. Notably, the high-linalool-titer strain (SA44) exhibited a distinct metabolic profile, with higher glycerol accumulation (~5.0 g/L). SA44 demonstrated superior sterol accumulation compared to SA49, with 1.6-fold higher squalene and 1.2-fold higher ergosterol production. Additionally, qRT-PCR was used to independently confirm the differential expression of seven regulatory genes, yielding a high Pearson correlation coefficient of 0.994. This study reports the first HAC1-mediated enhancement of linalool biosynthesis, establishing a robust strategy for microbial monoterpene production.