# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 3 Yeast Precursors Utilised
### Terpenoids
Similarly, the overexpression of the _ERG20_ gene encoding an enzyme with both dimethylallytransfersase and geranyltransferase activities and the sterol regulator _upc2-1_[60] has generally led to improved yields in recombinant terpenoid production. Improving the intracellular levels of the precursor molecule acetyl-CoA--an intermediate central to many metabolic pathways--has also been investigated. In one study by [PERSON] et al. [61], a _S. cerevisiae_ strain was developed by completely overhauling the native metabolic network involved in acetyl-CoA supply by incorporating several synthetic pathways which directly resulted in increased recombinant terpenoid levels. It was shown that by fusing mitochondria signals to sesquiterpenoid synthases, along with the introduction of a recombinant farnesyl diphosphate synthase, resulted in a marked increase of the citrus aroma compound valencene [62]. This demonstrates the prow  
Figure 3: Manipulations in mevalonate pathway for recombinant terpenoid production. Enzymes directly involved in the MVA pathway of _S. cerevisiae_ have been upregulated, downregulated or altered to increase the flux towards the production of various terpenoid-based aroma compounds. Additional recombinant enzymes needed for the catalysis of the terpenoid production are shown in orange with the respective aroma compounds being produced. Adapted from [53]. ERG10, acetyl-CoA C-acetyltransferase; ERG13, hydroxymethylglutaryl-CoA synthase; HMG1/HMG2, hydroxymethylglutaryl-CoA reductase 1/2; ERG12, mevalonate kinase; ERG8, Phosphomevalonate kinase; ERG19, mevalonate diphosphate decarboxylase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; IDII, isopentenyl diphosphate isomerase; ERG20, geranyl/ farnesyl diphosphate synthase; BTS1, GGPP synthase; ERG9, squalene synthase; crtYB, phytoene synthase and lycopene cyclase; crtl, crtE phytoene desaturase; geranylgeranyl diphosphate (GGPP) synthase. approaches where enzymes, substrates and intermediates are close to each other and competing pathways in the cytosol are avoided. Other successful attempts to produce terpenoids in yeast include the highly sought-after nootkatone, which imparts a strong grapefruit aroma [63]. This was achieved in _P. pastoris_ with a similar engineering strategy which has been proven fruitful for _S. cerevisiae_ to overexpression a truncated version of its _HMG1_ gene to improved levels. Carotenoids (specifically \(\beta\)-carotene) have also been produced in _S. cerevisiae_ by expressing carotenoid genes from strains from the ascomycete _Xanthophyllomyces dendrorhous_ (previously _Phaffia rhodozyma_) [64]. Increased concentrations of \(\beta\)-carotene were observed when the \(BTS1\) gene encoding a geranylgeranyl diphosphate synthase was overexpressed. These strains appear bright orange and although itself not an aroma compound, \(\beta\)-carotenes do serve as a substrate for carotenoid cleavage oxygenase, which releases compounds known as apocarotenoids. The expression of a carotenoid cleavage oxygenase from _Petunia hybrida_ in a strain already producing \(\beta\)-carotene, led to the release of detectable levels of a compound known as \(\beta\)-ionone, which has a highly desired violet scent [65]. Interestingly, a polycistronic version (genes separated from each other by viral T2A sequences) of the abovementioned carotenogenic genes was successfully expressed in yeast [66]. Assessing the feasibility of replacing elements of the MVA with that of the MEP pathway--theorized to yield a higher stoichiometric maximum plus having a lower requirement for oxygen--has been investigated. The yeast strain was developed where its MVA pathway was replaced with an MEP pathway. The resulting strain showed a slight growth defect made less biomass compared to the wild type, implying slight incompatibility [67].