# 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
Although the amounts of geraniol produced by this recombinant industrial yeast were much lower compared to the heterologous systems described above, the levels of this, as well as other monoterpenes, were way above the olfactory sensory thresholds, positively contributing to overall sensory characteristics of wines by providing enhanced floral and fruity attributes [45]. **Linalool** can be naturally found in many fruits and flowers and is responsible for pleasant floral aromas. Heterologous production of this flavour compound in yeast was originally achievedvia the expression of a linalool synthase (LIS) from _Clarkia breweri_, one of the earliest identified monoterpene synthases, and which specifically converts GPP to linalool [46; 47] (Figure 1). While initial concentrations of linalool were very low (~22 \(\upmu\)g/L), engineering of flux through the mevalonate pathway via overexpression of catalytic domain of Hmg1p (3-hydroxy-3-methylglutaryl coenzyme A reductase), combined with specific yeast strain selection, resulted in a 6-fold increase in linalool concentrations [48]. A subsequent study that used LIS from _Lavandula angustifolia_, combined with overexpression of _HMG1_ and downregulation of _ERG9_, resulted in levels of up to 95 \(\upmu\)g/L of linalool [49]. However, yields remain well behind those observed for geraniol and further optimization of this system is required if industrially-relevant production levels are to be achieved. As for geraniol, there have also been attempts to produce linalool in situ during fermentation instead of as a purified additive. Linalool and geraniol are the main impact molecules that confer dry-hopped flavour and aroma in specific styles of beer. In classical brewing, these monoterpenes originate from the flowers of the hop plant, which are added to the word during fermentation. In a recent study, [PERSON] et al. [50] screened for combination of linalool and geraniol synthases and gene promoters, that could be combined in brewing yeast to produce these monoterpenes at levels comparable to those achieved by standard dry-hopping during the beer fermentation process. A brewing strain of _S. cerevisiae_, that was metabolically engineered to produce higher levels of GPP, was equipped with a truncated LIS from _Mentha citrata_ and full-length GES from _Ocinum basilicum_, yielding a self-flavouring brewing yeast able to produce beer with basic dry-hopped flavour profiles, without the necessity of adding hop flowers. **Limonene** is another example of terpenoid that is commonly used as a flavouring agent [25]. It is mainly found in citrus fruit peels and used in food and beverage products such as candy and soft drinks [51]. With an annual production of over 60,000 tonnes [52], it is primarily obtained from citrus essential oils, which are byproducts from orange juice production, and may contain over 90% of this compound in the total pool of the compounds present [53; 54]. As the price of natural limonene is relatively high, its production in microbial hosts would reduce the dependence on its industrial production from plant sources, such as citrus fruits. The potential for heterologous production of limonene in _S. cerevisiae_ has recently been reported, where it is based on the same metabolic principals as those for the production of geraniol, except for the final step in which GPP is converted to limonene by a limonene synthase (LMS) (Figure 1). However, the reported yields of limonene remain far lower than those of either geraniol or lianool, with a maximum of 0.12 mg/L (+)-limonene and 0.49 mg/L (--)-limonene produced in _S. cerevisiae_[55; 56]. These low production levels suggest that major metabolic redesign and optimization are required for limonene production to be industrially applicable. However, as 27 limonene synthases, derived from 9 different plant families, have recently been characterized [57], this opens new possibilities for metabolic engineering and/or enzyme optimization in yeast, with potential to improve the yields of this valuable monoterpene.