# Heterologous Production of Flavour and Aroma Compounds in _Saccharomyces cerevisiae_
## 3 Heterologous Compounds Derived from Intermediates of the Mevalonate Pathway
### Isoprenoids
#### 3.1.2 Sesquiterpenes  
Sesquiterpenes are a diverse group of plant secondary metabolites which are generally derived from C-15 precursor; farnesyl pyrophosphate (FPP). **Valencene** can be found in many citrus fruits [61; 62] and is commonly and inexpensively extracted from the essential oil of Valencia oranges. Heterologous production of this sesquiterpene in _S. cerevisiae_ was achieved by identification and expression of valencene synthase (VS), with variants of this enzyme having been characterized from many plants including _Citrus paradisi_ (grapefruit), _Citrus sinensis_ or Nootka cypress (_Callitropsis nootkatensis_) [63; 64]. Expression of the _C. paradisi_ VS in a _S. cerevisiae_ strain (Figure 1), which was metabolically engineered to produce elevated levels of FPP [65], was shown to produce low levels of valencene (5.3 \(\mathrm{\SIUnitSymbolMicro g}\)/L) [66]. Later, [PERSON] and colleagues [63] have altered metabolic flux through the mevalonate pathway for enhanced farnesyl pyrophosphate (FPP) accumulation by overexpression of tHMG in _C. sinensis_ valencene synthase _CsTPS1_ expressing yeast strain, which led to 1.5-fold (ca. 40 to ca. 60 \(\mathrm{\SIUnitSymbolMicro g}\)/L) increase in valencene production, compared to the expression of _CsTPS1_ alone. Introduction either _A. thaliana_ short isoform of farnesyl diphosphate synthase (_At_FDPS), or _H. sapiens_ farnesyl diphosphate synthase (_Hs_FDPS), further increased valencene production; the final strain carrying _CsTPS1_, _tHMG_ and _AtFDPS_ was able to produce 370 \(\mathrm{\SIUnitSymbolMicro g}\)/L of valencene. Subsequent investigation of the effects of cellular compartmentalization have led to identification of the mitochondrion as a potential sub-cellular location for terpene production. To achieve this, the authors equipped _CsTPS1_ with mitochondrial targeting peptide from the _COX4_ gene, studied earlier by [PERSON] et al. [67], to test whether mitochondrial pool of FPP can be utilized for valencene production. Expression of mitochondrial _CsTPS1_ (_mtCsTPS1_) by itself resulted in nearly 3-fold increase in valencene production, followed by further 50% increase when co-expressed with _tHMG_, and additional 40% increase while mitochondria targeted FDPS (_mt_FDPS) was used, yielding ca. 1.2 \(\mathrm{mg}\)/L of valencene. Ultimately, the expression of an additional cytosolic copy of _CsTPS1_ resulted in production levels of approximately 1.5 \(\mathrm{mg}\)/L of this sesquiterpene, suggesting that both mitochondrial and cytosolic pools of FPP might be used for the production of terpen. In a recent study, the utility of exploring natural protein variation was highlighted by the discovery of an alternative valencene synthase derived from the heartwood of the Nootka cypress (CnVS) [64]. In comparison to valencene synthase obtained from _C. paradisi_, expression of CnVS in a non-optimized _S. cerevisiae_ strain produced 1.36 \(\mathrm{mg}\)/L of valencene, an almost 450-fold increase in production compared to the citrus equivalent and a level which approximates those achieved via extensive metabolic optimization of strains containing the citrus enzymes. This new valencene synthase may therefore represent a valuable resource for further optimization and industrial scale production of this sesquiterpene. **(+)-nootkatone** is an oxidized form of the sesquiterpene (+)-valencene, with a characteristic grapefruit flavour and relatively low sensory threshold [68]. It is found naturally in (and can be extracted from) plants including Citrus (_Rutaceae_ spp. ), Java (_Cyperus rotundus_), and Vetiver grass (_Vetiveria_ spp.) [69; 70]. Due to the very low concentrations of nootkatone found in natural sources, a synthetic compound is currently used for industrial applications [71]. However, as seen for many other flavour and aroma compounds, this synthetic production usually involves environmentally unfriendly processes and chemicals so heterologous production sources are being investigated [72; 73].