## 3 Results and Discussion  
### Overexpression of the Entire Mevalonate Pathway Genes in S. cerevisiae  
We began by creating strain FPPY005, which overexpresses all eight mevalonate pathway genes _ERG10_, _ERG13_, _tHMG1_ (encodes truncated HMGR), _ERG12_, _ERG8_, _ERG19_, _ERG20_, and IDII (Table 2, Figure 2). To do this, we constructed yeast expression cassettes for the mevalonate pathway genes with each construct containing two unique genes driven by the galactose-inducible bidirectional P\({}_{GAL1/10}\) promoter. Three copies of the gene encodingtruncated HMGR (_tHMG1_) are integrated because the enzyme was previously shown to be rate-limiting [21]. We opted to use the native terminator for each gene (i.e., _ERG10_ terminator was used for _ERG10_ ORF) to minimize the number of fragments required for homologous recombination. _URA3_ selectable marker was chosen as it allows for simple recycling of the marker via loxP-Cre recombination and counter-selection with 5\({}^{\prime}\)-fluoroorotic acid (5\({}^{\prime}\)-FOA) [22]. The gene cassettes were placed in the shuttle vector (pRSII416) for ease of cloning.  
The gene expression cassettes for the upregulation of the mevalonate pathway were sequentially integrated into the genome of BCC39850 hlu (_S. cerevisiae_ BCC39850 with triple deletions) at designated sites, as shown in Figure 2 (_YPRCA15c_, _ARS308a_, _ARS1021_, etc.). Integration of gene expression cassettes at these sites has previously been shown to lead to high gene expression levels [23].  
The integration of the five gene expression cassettes should lead to the overexpression of all eight genes in the mevalonate pathways, namely _ERG10_, _ERG13_, _tHMG1_ (encodes truncated HMGR), _ERG12_, _ERG8_, _ERG19_, _ERG20_ and _IDI1_. To verify this, we performed real-time PCR (RT-PCR) in the engineered strain FPPY005_39850 and the parental strain BCC39850 hlu under inducing (i.e., galactose-containing medium) and non-inducing (i.e., glucose-only medium) conditions (Figure 3). Real-time PCR analysis indicates that all mevalonate pathway genes are up-regulated under inducing conditions (Figure 3A). As expected (given the two additional copies of the _tHMG1_ gene expression construct), the strongest up-regulation was observed in the expression of the truncated HMGR in strain  
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FPPY005_39850, where we saw 11.5-fold overexpression compared to levels observed in non-inducing conditions (i.e., medium containing glucose as the sole carbon source; (relative expression levels in galactose medium))/(relative expression levels in glucose medium) (Figure 3A). The lowest up-regulation was observed in the expression of IDI1, at 2.5-fold overexpression compared to levels observed in non-inducing conditions. We did not observe any increase in the expression levels of mevalonate pathway genes in the parental strain BCC39850 hlu under inducing conditions (Figure 3B). Accumulatively, these results indicate that the integration of the mevalonate pathway gene expression cassettes led to the upregulation of the mevalonate pathway in strain FPPY005_39850.  
Figure 3: Real-time PCR (RT-PCR) analysis of mevalonate pathway genes in engineered strains FPPY005_39850 (**a**) and BCC39850 hlu (**b**).  
Figure 2: Pathway design for the construction of FPP-overproducing _S. cerevisiae_ strain. Block arrows designate mevalonate pathway genes; block squares are endogenous terminators; thin arrows are endogenous promoters; grey rectangles are native auxotrophic selectable markers (the URA3 selectable marker is flanked by loxP sites). The integration sites (_YPRCA15c_, _ARS308a_, _ARS1021_, _ARS720u_, and _ARS1309_) for the gene constructs are marked.