# Heterologous Production of Flavour and Aroma Compounds in _Saccharomyces cerevisiae_
## 3 Heterologous Compounds Derived from Intermediates of the Mevalonate Pathway
### Isoprenoids
#### 3.1.1 Monoterpenes  
Monoterpenes are all produced via the action of monoterpene synthases acting on geranyl pyrophosphate (GPP) as a common substrate. Each individual monoterpene synthase is therefore responsible for producing a specific monoterpene (or a combination of monoterpenes) from this common metabolic precursor [31]. **Geraniol** is one of the most in-demand monoterpenes, primarily due to its pleasant rose-like aroma. It is now an established additive in the food and beverage industries, where it is commonly used to enhance the flavours of a broad range of beverages, ice cream, candy, chewing gum, and many other products [32]. Due to its high sensory impact, geraniol is produced in negligible concentrations in most plants, such that the yields of final extracted compounds are very low [33]. While plant sources of geraniol are generally limiting, yeast has proven to be a very efficient cell factory for the heterologous production of this compound. _S. cerevisiae_ lacks the ability to produce monoterpenes, however it produces the penultimate monoterpene precursor, GPP, as part of the mevalonate/ergosterol pathway [34; 35] (Figure 1). Interestingly, despite lacking recognised monoterpene synthase activity, _S. cerevisiae_ strains carrying mutations in farnesyl diphosphate synthase gene (_ERG20_) were shown to produce low levels of geraniol (1.3 mg/L), presumably through non-specific production due to high GPP levels [36]. Later, [PERSON] and colleagues isolated and characterized the specific geraniol synthase (GES) from sweet basil (_Ocinum basilicum_) that catalyses conversion of GPP to geraniol [37], unlocking the possibility of geraniol production in microbial hosts (Figure 1). Heterologous expression of this enzyme in _S. cerevisiae_ allowed production of geraniol at levels up to 500 \(\upmu\)g/L in the growth medium [34]. Following on from this, engineering of ERG20p via amino acid substitutions surrounding the catalytic site resulted in a strain able to produce around 5 mg/L of geraniol [38]. Geraniol synthase enzymes have now been identified and characterized from a large number of different plant sources [39; 40]. This expanded catalogue of natural variation, combined with additional metabolic engineering, has been used to further improve heterologous geraniol production in _S. cerevisiae_. By introducing a fusion protein composed of geraniol synthase from _Valeriana officinalis_ (tVoGES) that lacks plastid targeting signal and a mutant farnesyl diphosphate synthase ERG20p[P96W-N127W], in a strain with a metabolically-engineered high-flux mevalonate pathway, 239 mg/L of geraniol was achieved in fed-batch cultures [41; 42; 43]. However, this concentration was still deemed inadequate for industrialization of the system. In an elegant study [PERSON] and colleagues [44] have now achieved much higher levels of geraniol production through a combination bioprospecting for highly active GES proteins, site directed mutagenesis and enzyme structural analysis, which ultimately resulted in a strain able to produce over 0.5 g/L of geraniol. When this strain was further tested in fed-batch fermentation under carbon restricted conditions, it produced 1.68 g/L of geraniol, the highest level of this monoterpene produced by any eukaryotic organism reported to date [44]. In parallel to approaches that seek to produce the highest amounts of geraniol possible for purification as an additive, alternative studies have been conducted to investigate the in situ production of geraniol during wine fermentation [45]. Expression of GES from sweet basil resulted in the accumulation of more than 750 \(\upmu\)g/L of geraniol in finished wine that was made from aromatically neutral grapes. Surprisingly, in additional to geraniol, the final wine also contained significant amounts of additional monoterpenes, including citronello,linalool, and nerol, which were likely derived from indigenous enzymatic activities in the industrial wine yeast background.