# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 3 Yeast Precursors Utilised  
### Phenylpropanoids  
The aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan serve as the precursors to many compounds of commercial interest [27]. More specifically, L-phenylalanine, L-tyrosine provide the precursors for a large group of compounds called phenylpropanoids--of which many have aroma-active properties. The biosynthesis of the aromatic amino acids proceeds via the shikimate pathway [28] (Figure 2). It is a seven-step metabolic pathway leading to the production of chorismate, the common aromatic precursor to all three amino acids. The shikimate pathway is initiated with the condensation of phosphoenolpyruvate (PEP)--an intermediate in the glycolysis pathway--and erythrose-4-phosphate (E4P)--an intermediate in the pentose phosphate pathway--to generate 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). Chorismate is the branching node, where L-tryptophan is separated from the other two amino acids as chorismate is converted to prephenic acid (the precursor molecule of L-phenylalanine and L-tyrosine) by a chorismate mutase. Subsequent decarboxylation and transamination events lead to the production of L-tyrosine and L-phenylalanine. In general, intracellular L-tyrosine levels in _S. cerevisiae_ are about ten-fold higher than L-phenylalanine with L-tryptophan 10 times less than L-phenylalanine [29]. _S. cerevisiae_ has a limited capacity to process aromatic amino acids beyond using them for protein synthesis. Pathways involved in using L-tyrosine and L-phenylalanine as precursors have been  
Figure 2: Biosynthetic pathway for phenylpropanoids. Yeast can synthesize all three aromatic amino acids (L-phenylalanine, L-tyrosine and L-tryptophan) via the shikimate pathway but have few processing capabilities beyond utilising them in peptide synthesis or their catabolism via the Ehrlich pathway (which can produce the aroma compound 2-phenylethanol). _S. cerevisiae_ and other yeast have been exploited to convert their free aromatic amino acids to compounds with aroma properties. The recombinant enzymes that have been incorporated in yeast to convert precursors to aroma compounds of commercial value are shown below each recombinant metabolite. incorporated into yeast to produce a multitude of compounds and of these, the phenyl ring structure represents a central feature (Figure 2). A key aroma compound derived from the shikimate pathway is that of vanillin (imparting vanilla flavour) and has been the subject of many investigations in the past due to its high value and wide use. Vanillin is not synthesised from any of the aromatic amino acids, but from an intermediate in the shikimate pathway, namely dehydrohikimate. The first report of vanilla production by yeast used three recombinant genes in the fission yeast _S. pombe_ to transform dehydrohikimate to vanillin [30]. In the same study, _S. cerevisiae_ was also used, but an additional activation enzyme was needed. Vanillin is moderately toxic to yeast cells (it represses translational processes [31]). It was shown that adding a glycosyl moiety, by expressing a 1-UDP-glycostransferase, leads to the conversion of vanillin to vanillin-glucoside (VG), which markedly increased production levels. Remarkable improvements in VG titres have been achieved with rational engineering design approaches: in silico metabolic engineering algorithms have been implemented to identify yeast target genes that could enhance productivity [32]. Manipulations of two of the identified targets (_PDC1_ and _GDH1_) led to a five-fold improvement of VG yields and was attributed to the recycling of the supply of cofactors. Additional modelling-based methodologies underlined the utility of in silico design for improvement in VG levels [33; 34]. The pathway for the production of _p_-hydroxycinnamic acid (also known as _p_-coumaric acid), which imparts a cinnamon aroma, has been incorporated in _S. cerevisiae_[35].