# Heterologous Production of Flavour and Aroma Compounds in _Saccharomyces cerevisiae_
## 2 Heterologous Compounds Derived from Intermediates of the Shikimate Pathway
As there are hundreds of different strains of _S. cerevisiae_ available, this work clearly demonstrates that the original genetic background is highly important in regards of choosing a yeast strain for an efficient microbial cell factory. **Raspberry ketone** is the primary compound that contributes to the aroma of raspberries (_Rubus idaeus_) ([PERSON], 1998; [PERSON], 1998), although it is also produced by many other plants, including cranberries, blackberries, and rhubarb ([PERSON], 1998). Raspberry ketone extracted from natural sources is an expensive flavoring agent, costing as much as US $3000/kg. However, as seen for vanillin, raspberry ketone can also be derived utilizing chemical synthesis, with the synthetic version costing far less than the naturally-derived version (US $58/kg) ([PERSON], 1998). In _R. idaeus_, raspberry ketone is produced via the diketide pathway, starting with condensation of 4-counaryl-CoA and malonyl-CoA by benzalacetone synthase (BAS) to form 4-(4-hydroxyphenyl)but-3-en-2-one (benzalacetone) ([PERSON], 1998; [PERSON], 1998). In a subsequent reaction, benzalacetone is reduced by NADPH-dependent benzalactone reductase (BAR), also known as raspberry ketone/zingerone synthase 1 (_Ri_ZS1), to form raspberry ketone ([PERSON], 1998). Figure 1. Simplified central metabolism of _Saccharomyces cerevisiae_ (blue arrows), and heterologous enzymatic reactions (green arrows) leading to production of various flavour and aroma compounds: vanillin-glucoside (3 DSD: 3-dehydrooshikimate dehydratase, ACAE: aryl carboxylic acid reductase, OMT: O-methyltransferasefaserase, UGT: UDP-glycosyltransferasezeszes), raspberry ketone (PAL: phenylalanine/tyrosine ammonia lyase, CCH: cinnamate-4-hydroolase, 4 CL2: oumarate-CoA ligase 2, BAS: benzalacetone synthase), cinnamaldehyde (PAL2: phenylalanine ammonia lyase 2, ACAE: aryl carboxylic acid reductase, EntD: phosphoplentheynethenyl transferase), geranoid (GES: geranoid synthase), limonene (LMS: limonene synthase), linalool (LIS: linalool synthase), sabinene (SAS: sabinene synthase), valence (VS: valencene synthase, mFlPs: mitochondria targeted valence synthase), (+)-nootkatone, _trans_-nootkatol, _cis_-nootkatol (VOX: (+)-valence oxidase), nerolled (NES: nerolled synthase), \(\beta\)-iononene (crtyB: phytoene synthase/lycopene cyclase, crtl: phytoene desurase, CCD: carotenoid cleavage dicorygenesis), stevieid glycosides (CDPS: copalyl diphosphate synthase, KS: kaurene synthase, KO: kaurene oxidase, KAH: kaurencic acid-13-hydroxylase, UGT: UDP-glycosyltransferase). S. cerevisiae lacks the diketide pathway, with phenylalanine and tyrosine representing the metabolic branch points for heterologous de novo production of RK (Figure 1). Primary attempts of heterologous expression of plant genes, 4-coumarate-coenzyme A ligase (4 CL) and chalcone synthase (CHS), in this yeast for raspberry ketone production were not entirely successful. The recombinant yeast grown in media containing \(p\)-coumaric acid produced only very low levels of raspberry ketone [19]. However, in a recent study, [PERSON] et al. [14] engineered _S. cerevisiae_ to express the complete de novo pathway for heterologous production of raspberry ketone, without necessity of supplementation with the precursor; \(p\)-coumaric acid. The recombinant yeast strain was genetically modified to produce \(p\)-coumaric acid through the expression of the heterologous enzymes phenylalanine/tyrosine ammonia lyase from _Rhodosporidium toruloides_ (_Rt_PAL) (which provided both phenylalanine and tyrosine ammonia lyase activists), and cinnamate-4-hydroxylase from _A. thaliana_ (_A_t_C4H), a combination shown previously to drive \(p\)-coumaric acid production in _S. cerevisiae_[20; 21]. Additional expression of a synthetic fusion protein that linked coumarate-CoA ligase 2 from _Petroselinum crispum_ and benzalacetone synthase from _Rheum palmatum_ (_Pc_4 CL2-_Rp_BAS), yielded a strain able to produce 3.5 mg/L of raspberry ketone [14].