# 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
Conversion of (+)-valencene to its oxidation products; (+)-nootkatone and \(\alpha\)- and \(\beta\)-nootkatole (all having pleasant grapefruit-like aromas) are thought to be catalysed by members of the cytochrome P450 monoxygenase superfamily. Co-expression of the P450 enzyme CYP71 AV8 from chicory root (_Cichorium intybus_), also known as (+)-valencene oxidase (VOX), which was reported to convert valencene to nootkatone via its oxidation at the C2 positions, along with valencene synthase from _C. sinensis_, allowed a recombinant _S. cerevisiae_ strain to convert 68% of total pool of (+)-valencene to _trans_-nootkatol (0.92 \(\mathrm{mg}\)/L), 8% to _cis_-nootkatol (0.11 \(\mathrm{mg}\)/L), and 3% to (+)-nootkatone (0.04 \(\mathrm{mg}\)/L) [74] (Figure 1). In a later study, [PERSON] et al. [75] used an in silico gene mining approach to identify and characterize novel cytochrome P450 valencene 2-oxygenases; CYP71D4 from _Solanum tuberosum_, CYP71D51v2 from _Nicotiana tabacum_, CYP71D1 from _Catharantthus roseus_, and CYP71D326 fromRicinus communis. Subsequently, they tested each of these oxygenases by introducing them into WAT11 yeast strain, which was genetically modified for stable expression of _A. thaliana_ P450 reductase _ATR1_[76]. The tobacco (_Nicotiana tabacum_) enzyme, CYP71D51v2, displayed the highest expression levels in this recombinant yeast, as well as efficient conversion of (+)-valencene to \(\beta\)-nootkatol and low levels of undesired byproducts. Incubation of this yeast strain with increasing (+)-valencene concentrations resulted in maximum combined yield of 6 mg/L of \(\beta\)-nootkatol and (+)-nootkatone, while 200 mg/L (+)-valencene was used. Further increase of (+)-valencene feed concentrations resulted in lower conversion rates, likely due to increased substrate and/or product toxicity. Production of _trans_-nootkatol was further improved by overexpression of the native yeast type III membrane associate protein Ice2p [77]. Introduction of this genetic modification into recombinant strain engineered earlier to produce (+)-valencene via expression of premnspirodiene oxygenase (HPO; CYP) from _Hyoscyamus muticus_ and cytochrome P450 from _A. thaliana_ (_At_CPR) [64], resulted in stabilizing the levels and enzymatic activity of the latter heterologous enzyme. This modified recombinant strain was able to synthesize approximately 30 mg/L of _trans_-nootkatol. Although promising, the heterologous nootkatone production system would need to be further improved for its potential application on the industrial scale. **Nerolidol** occurs in a broad spectrum of plants and, in its purified form, displays wood- and fresh bark-like aromas. It is officially permitted to be used as food flavouring additive by the U.S. Food and Drug Administration (FDA) and is currently used in industrial processing [25]. Recently, a study has explored the heterologous production of this sesquiterpene by _S. cerevisiae_ via the expression of a nerolidol synthase (NES) from _Actinida chinensis_ in an _ERG20_ overexpression background, resulting in the production of nerolidol in the growth media at 5 mg/L [78]. Further pathway optimization of this strain, which included replacing the initial three enzymes of \"upper\" MEV pathway (_ERG10, ERG13_, and _tHMG1_) with codon optimized heterologous counterparts and co-expressing HMG-CoA synthase and a bi-functional acetoacetyl-CoA thiolase/HMG-CoA reductase from _Enterococcus faecalis_ resulting in over a ten-fold increase in nerolidol (56 mg/L). The authors also addressed the issue of undesired production of high levels of squalene, resulting from enzymatic activity of squalene epoxidase (_ERG9_), which competes with the heterologous nerolidol synthase (_Ac_NES1) for the cellular pools of FPP.