## 3 Results and Discussion
### Improving Terpenoid Production via Downregulation of ERG9 Gene Expression  
In order to improve the production titer of the fragment terpenoids, we explored two complementary strategies: (1) down-regulation of _ERG9_ expression and (2) fusing the FPP synthase and the terpene synthase enzymes. In the first strategy, _ERG9_ encodes squalene synthase, which catalyzes the first committed step of ergosterol biosynthesis (Figure 1). Since the enzyme uses FPP as a substrate, downregulation of its expression was hypothesized to divert the metabolic flux away from the production of ergosterol to the product of interest. In previous works, _ERG9_ downregulation has led to improvement in the production of several high-value terpenoids such as artemisinin, casbene, and lycopene [21, 24, 25]. In order to downregulate _ERG9_ expression, we used the CRISPR/Cas9 system to replace the endogenous _ERG9_ promoter with an L-methionineressible promoter from _MET3_ (PMET3) in strain FFPY005_39850, resulting in strain FFPY005_39850 _PMET3_-_ERG9_. In parallel, we also created an analogous strain named WWY005_PMET3_-_ERG9_, which has the same genetic modifications as strain FFPY005_39850 _PMET3_-_ERG9_[15]. The only difference between the two strains is that WWY005_PMET3_-_ERG9_ is derived from the laboratory strain CEN.PK2-1C while strain FFPY005_39850 _PMET3_-_ERG9_ is derived from strain BCC39850. We transformed strains WWY005_PMET3_-_ERG9_ and WWY005 with the plasmid over-expressing _CITPS2_ and measured the production of \(\alpha\)-santalene by the resulting strains in media either with or without L-methionine supplementation (Figure 6). Without L-methionine supplementation, strain WWY005_PMET3_-_ERG9_ harboring the _CITPS2_ overexpression plasmid produced \(\alpha\)-santalene at a titer of 12.1 \(\pm\) 0.1 mg/L, a 94% improvement in titer compared to the level observed in strain WWY005 harboring the same _CITPS2_ overexpression plasmid (6.3 \(\pm\) 0.2 mg/L). The titer of \(\alpha\)-santalene was further improved to 17.3 \(\pm\) 0.1 mg/L when 1 mM L-methionine was supplemented to the fermentation medium. Our results suggest that _ERG9_ downregulation is a viable strategy for improving fragment terpenoid production. Figure 5: 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 of strains FFPY005_39850 harboring pRSII426-Gal1/10-AcHS1 (**B**) and FFPY005_39850 harboring the empty vector pRSII426-Gal1/10 (**C**). We obtained an even more dramatic improvement in \(\alpha\)-humulene production when _ERG_9 was repressed (Figure 7). Specifically, without L-methionine supplementation, strain FPPY005 P\({}_{\text{MET3}}\)_-ERG_9 harboring the _AcHS1_ overexpression plasmid produced \(\alpha\)-humulene at a titer of 94.1 \(\pm\) 3.3 mg/L, a 5.0-fold improvement in titer compared to the level observed in strain FPPY005 harboring the same _AcHS1_ overexpression plasmid (18.7 \(\pm\) 0.8 mg/L). Interestingly, the addition of 1 mM L-methionine to the production media did not further increase \(\alpha\)-humulene titer (as was the case for \(\alpha\)-santalene production) but rather decreased the titer to 78.1 \(\pm\) 5.3 mg/L, a 17% decrease compared to no additional L-methionine supplementation. Accumulatively, these results underscore the importance of fine-tuning the level of _ERG_9 repression. Moreover, similar results in both strains (FPPY005_39850 P\({}_{\text{MET3}}\)_-ERG_9 and WVY005 P\({}_{\text{MET3}}\)_-ERG_9) suggest that the strategy of _ERG_9 repression to improve terpenoid production is general and may not be specific to the background strain. Figure 6: Effects of _ERG_9 repression on \(\alpha\)-santalene production in engineered strains. + indicates that the gene was overexpressed. – indicates that the gene was not overexpressed. N.D. not detected. Figure 7: Effects of _ERG_9 repression on \(\alpha\)-humulene production in engineered strains. + indicates that the gene was overexpressed.