# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 3 Yeast Precursors Utilised
### Terpenoids  
Terpenoids (also called terpenes or isoprenoids) are the largest and most diverse group of natural compounds. They are derived from the basic five-carbon (C5) precursor unit isopentenyl diphosphate (IPP) and its double-bond isomer dimethylallyl diphosphate (DMAPP) that can be assembled and modified in over 60,000 different types of terpene-like structures. Apart from the exceptional flavour qualities of many terpenoids, certain terpenoids have promising applications in biofuel and antimicrobial research [48]. Terpenoids are either produced via the mevalonate biosynthesis pathway (MVA) or the 2-C-methyl-D-erythritid-4-phosphate pathway (MEP) with the former being the best-studied and found in yeast. In this pathway, acetyl-CoA is condensed to produce the universal isoprene building unit (C5), isopentenyl diphosphate (IPP). Subsequent condensations conducted by prenyltransferases of IPP and DMAPP result in terpenoid precursors called polyisoprenoid diphosphates of different lengths: geranyl diphosphate (GPP) for monoterpenoids (C10), farnesyl diphosphate (FPP) for sesquiterpenoids (C15), geranylgeranyl diphosphate (GGPP) for triterpenoids (C20), 2 units of FPP for triterpenoids (C30) and 2 units of GGPP for tetraterpenoids (C40). The C30 and C40 precursors lead to the biosynthesis of sterols and carotenoids, respectively. Cyclisation of the abovementioned polyisoprenoid diphosphates are catalysed by a large group of enzymes called terpene synthases to generate terpenoids with single or multiple ring structures (with some remaining open). These enzymes often display a high level of promiscuity with regards to their substrate preference leading to the large diversity among terpenoid structures. In addition, tailoring enzymes like oxygenases, methyltransferases, acetyltransferases and glycosyltransferases can add functional groups to different positions of the terpenoid structure. As mentioned, yeast including _S. cerevisiae_, do possess an MVA pathway but does not have terpene synthases that are able to produce monoterpenoids, sesquiterpenoids and diterpenoids. _S. cerevisiae_ implements the MVA pathway to produce sterols (specifically ergosterol) that are structural elements of the cell membrane and impart modulation to the membrane fluidity. It is an essential pathway for yeast as strains with mutated genes in this pathway require exogenous sterol for survival [49]. Significant efforts have been made to create a yeast platform that would be able to produce terpenoids as discussed in multiple papers and reviews [50; 51; 52; 53; 54] (Figure 3). A yeast without any modification within its MVA pathway would produce negligible levels of recombinant terpenoids. A key strategy to improve levels would be to direct carbon flux away from producing sterols without the complete elimination of the pathway. On a transcriptional level, the native promoter of the squalene synthase _ERG9_ gene (that encodes the enzyme that catalyses the first reaction of converting famesyldiphosphate to ergosterol) was replaced with repressible promoters which led to subsequent lower concentrations of ERG9, facilitating the increased levels of recombinant terpenoid production [55]. Recently, a degradation tag attached to ERG9 was shown to destabilise the protein, which also led to a dramatic improvement in recombinant terpenoid production without compromising the cell viability to any significant extent [56]. The overexpression of a truncated version of the hydroxymethylglutaryl-CoA reductase (tHMG1), that is devoid of its transmembrane moiety and is thus present in the cytosol, led to an increased amount of squalene. This confirmed that HMG-CoA reductase, the enzyme that produces mevalonate, is a rate-limiting step [57]. Many subsequent attempts using yeast to produce recombinant terpenoids contain this feature [58; 59].