## 3 Results and Discussion
### Screening of Terpene Synthases in FPPY005_39850  
Having verified that the mevalonate pathway is up-regulated in strain FPPY005_39850, we next turned our attention to identifying efficient terpene synthase enzymes for the production of fragrant agarwood and sandalwood terpenoids in the context of yeast. Specifically, we screened seven distinct terpene synthases from agarwood, sandalwood, and related species with previously reported enzymatic activities [5]. These include three sesquiterpene synthase genes (_AsSeTSPS_, _AsSeTSPS1_ and _AsASSI_) from _Aquilaria sinensis_, \(\alpha\)-humulene synthase gene (_AcHS1_) from _Aquilaria crassua_, \(\delta\)-guaiene synthase gene (_AmdGS1_) from _Aquilaria microcarpa_, \(\beta\)-eudesmol synthase gene (_ZzBES2_) from _Zingiber zerumbet_ and santalene synthase gene (_CITPS2_ or _CSanSyn_) from _Clusena lansium_ (Table 2). We constructed the gene cassettes for the expression of seven distinct terpene synthase genes. For these gene expression cassettes, we used codon-optimized and synthesized genes to ensure robust expression in yeast. As with the mevalonate gene expression cassettes, we used the galactose-inducible bidirectional PGAL1/10 promoter to drive the expression of the terpene synthase genes. However, we used the T\({}_{\text{HISS}}\) terminator as the universal terminator. The gene expression cassettes were placed in the shuttle vector (pRSII426), which contains a 2 micron origin of replication, to ensure that the plasmid is maintained in the cell at a high-copy number.  
We transformed strain FPPY005_39850 with plasmids harboring the individual terpene synthase expression cassette and measured the production of fragrant terpenoids using GC-MS (for product identification) and GC-FID (for product quantification). To minimize the evaporation of the fragrant terpenoids, the cultures were overlaid with 20% dodecane. Gratifyingly, we observed the production of several fragrant terpenoids including \(\alpha\)-humulene, \(\delta\)-guaiene, and a small amount of \(\alpha\)-guaiene from agarwood and \(\alpha\)-santalene from sandalwood in our engineered strains (Figures 4, 5 and S1-S3). For the production of agarwood terpenoids, FPPY005_39850 overexpression _AsSeTSPS1_ produced \(\alpha\)-humulene at a titer of 14.6 \(\pm\) 0.4 mg/L (Figure 4), while the strain overexpressing _AcHS1_ produced \(\alpha\)-humulene at a titer of 22.3 \(\pm\) 0.7 mg/L (Figure 5). FPPY005_39850 overexpression _AmdGS1_ produced \(\delta\)-guaiene at a titer of 14.5 \(\pm\) 0.7 mg/L and a trace amount of \(\alpha\)-guaiene (Figure S1). FPPY005_39850 overexpressing _ZzBES2_ produced \(\beta\)-eudesmol at a titer of 16.5 \(\pm\) 0.5 mg/L (Figures S3-S4). Importantly, to our knowledge, this is the first demonstration of \(\beta\)-eudesmol production in the yeast system. For the production of sandalwood terpenoids, the engineered strain overexpressing CITPS2 produced \(\alpha\)-santalene at a titer of 6.3 \(\pm\) 0.2 mg/L.  
Figure 4: Total ion chromatograms (left panel) and mass spectra (right panel) obtained from GC-MS analysis of an authentic standard for \(\alpha\)-humulene (**A**) and dodecane samples from the cultivation ofstrains FFPY005_39850 harboring pRSII426-Gal1/10-AsesTPS1 (**B**) and FFPY005_39850 harboring the empty vector pRSII426-Gal1/10 (**C**).