## 3 Results and Discussion
### Improving Fragment Terpenoid Production via Enzyme Fusion
For \(\alpha\)-santalene production, additional expression of FPPS led to a 32.6% increase in \(\alpha\)-santalene titers compared to the strain overexpressing _C_/TPS2 alone (37.9 \(\pm\) 2.8 mg/L vs. 28.5 \(\pm\) 1.7 mg/L) (Figure 8a). The expression of FPPS and _C_/TPS2 as fusion enzymes using the GGGGS linker further improved the production titer to 47.6 \(\pm\) 0.1 mg/L. This is a 66.9% increase over the strain overexpressing _C_/TPS2 alone and a 25.8% increase over the strain overexpressing FPPS and _C_/TPS2 as distinct enzymes. Interestingly, expression of the FPPS-GSG-C_/TPS2 fusionenzymes lowered \(\alpha\)-santalene production when compared to the strain overexpressing FPPS and _C_ITPS2 as distinct enzymes. For \(\delta\)-guaiene production, additional expression of FPPS did not increase \(\delta\)-guaiene titer compared to the strain overexpressing _Amd_GS1 alone (40.2 \(\pm\) 5.1 mg/L and 42.1 \(\pm\) 7.5 mg/L, respectively) (Figure 8b). However, the expression of FPPS and _Amd_GS1 as fusion enzymes improved the production titer to 54.5 \(\pm\) 5.5 mg/L (for the GSG-linked fusion enzyme) and 52.9 \(\pm\) 3.3 mg/L (for the GGGGS-linked fusion enzyme), equivalent to approximately 30% increase in titer. For the remaining two terpenoid targets, \(\alpha\)-humulene and \(\beta\)-eudesmol, enzyme fusion did not improve the production titer in a meaningful way. For \(\alpha\)-humulene production, the biggest improvement in production titer came from increased expression of FPPS as a standalone enzyme. Specifically, the strain with additional expression of FPPS led to a 20.3% increase in \(\alpha\)- humulene titers compared to the strain overexpressing _Ac_HS1 alone (94.0 \(\pm\) 1.1 mg/L vs. 78.1 \(\pm\) 5.3 mg/L) (Figure 8c). The expression of FPPS and _Ac_HS1  
Figure 8: Improving fragment terpenoid production via enzyme fusion. (**a**) Production of \(\alpha\)-santalene in engineered strains overexpressing _C_ITPS2 either alone or as fusion enzymes with FPPS; (**b**) Production of \(\delta\)-guaiene in engineered strains overexpressing _Amd_GS1 either alone or as fusion enzymes with FPPS; (**c**) Production of \(\alpha\)-humulene in engineered strains overexpressing _Ac_HS1 either alone or as fusion enzymes with FPPS; (**d**) Production of \(\beta\)-eudesmol in engineered strains overexpressing _Z_zBES2 either alone or as fusion enzymes with FPPS. N.D. not detected. as fusion enzymes using either the GSG or GGGGS linker did not significantly improve the production titer of \(\alpha\)-humulene (93.2 \(\pm\) 5.1 mg/L and 101.7 \(\pm\) 6.9 mg/L, respectively). The most surprising results came from the strains producing \(\beta\)-eudesmol (Figure 8d). Additional expression of FPPS led to a 61.4% decrease in \(\beta\)-eudesmol titers compared to the strain overexpressing \(Z\)zBES2 alone (9.2 \(\pm\) 1.2 mg/L vs. 23.9 \(\pm\) 1.1 mg/L). We were able to revert some of the titer loss by employing the enzyme fusions. Specifically, the expression of FPPS and \(Z\)zBES2 as fusion enzymes using the GSG and GGGGS linkers resulted in \(\beta\)-eudesmol titers of 21.1 \(\pm\) 0.2 mg/L and 12.3 \(\pm\) 0.7 mg/L, respectively. Overall, our engineered strains produce fragment terpenids at similar levels to previous reports [45; 46; 47; 48]. For example, [PERSON] and coworkers engineered the yeast _S. cerevisiae_ to produce \(\alpha\)-humulene by targeting both the mevalonate pathway enzymes and the \(\alpha\)-humulene synthase to the yeast peroxisome, resulting in an engineered strain (M4-Ase) that produced \(\alpha\)-humulene at a titer of 76.16 mg/L and 144.91 mg/L in glucose- and galactose-containing medium, respectively [46]. [PERSON] and coworkers engineered the yeast _S. cerevisiae_ to produce \(\delta\)-guaiene, \(\alpha\)-guaiene, and (\(-\))-rotundone (an oxidized product of \(\alpha\)-guaiene) [49]. The authors reported the titer of only (\(-\))-rotundone (\(-\)1.2 mg/L) but not the other two compounds. A similar effort to produce \(\delta\)-guaiene in the bacterium _E.