# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 3 Yeast Precursors Utilised
### Phenylpropanoids
This simply involved the incorporation of various phenylalanine ammonia-lyases (PAL)/tyrosine ammonia-lyases (TAL) which deaminase L-tyrosine. Several metabolic engineering strategies have proven successful in enhancing _p_-hydroxycinnamic acid along with the levels of so-called _trans_-cinnamic derivatives (which include cinnamaldehyde, cinnamyl alcohol and hydrocarbonl) [36]. These strategies involved removing known feedback-regulated steps of aromatic amino acid biosynthesis and directing the flux towards the production of these _trans_-cinnamic compounds by side-tracking the decarboxylation step of the competing Ehrlich pathway. A phenylacrylic acid decarboxylase (PAD1) is thought to be responsible for the decarboxylation of _trans_-cinnamic derivatives, as a _pad1_ knockout strain showed no endogenous activity on trans-cinnamic acid and _p_-hydroxycinnamic acid [37]. Nevertheless, the _trans_-cinnamic derivatives are converted to less toxic compounds by the yeast via unknown mechanisms [36]. It was found, similar to vanillin, that by adding a glycosyl moiety to trans-cinnamic acid catalysed by an UDP-glucose:cinnamate glucosyltransferase reduces its toxicity and led to increased levels. A recent addition to the phenylpropanol aroma compounds that are recombinantly produced in yeast is that of raspberry ketone [4-(4-hydroxyphenyl)butan-2-one] [38]. This involved the incorporation of a four-gene pathway from various organisms into yeast that converted L-phenylalanine and L-tyrosine to raspberry ketone. Testing various enzyme combinations and fusions resulted in higher levels of raspberry ketone. Improving yields of 2-phenylethanol (2-PE)--a compound with a rose-like aroma--has been investigated extensively. 2-PE is the fusel alcohol of L-phenylalanine and of the four phenylpropanol aroma compounds discussed, 2-PE does not require the expression of recombinant genes as it arises from the catabolism of L-phenylalanine via the Ehrlich pathway. This includes its deamination, decarboxylation and reduction that are conducted by ARO9, ARO10 and various alcohol dehydrogenases (ALD1-5) in _S. cerevisiae_ respectively. Metabolic engineering efforts to increase 2-PE levels included the streamlining the Ehrlich pathway which involved the overexpression of _ARO9_ and _ARO10_ with the concomitant removal of a competing phenylacetaldehyde oxidase (_ALD3_) [39]. A transcription factor (ARO80) is known as an activator of the _ARO9_ and _ARO10_ genes and its overexpression, together with _ARO9_ and _ARO10_, led to a four-fold increase in 2-PE levels. Efforts have also been made to increase the intracellular levels of the precursors of the shikimate pathway PEP and E4P. Especially targeting E4P, which has lower intracellular concentrations than PEP [40] would result in an equal balance of the two precursors and could facilitate improved flux toward aromatic amino acid production. Attempts thus far to increase levels of E4P have involved alternations within the pentose phosphate pathway [41]. It was shown that the deletion of the glucose-6-phosphate dehydrogenase (_ZWF1_) gene and overexpression of the transketolase (_TKL1_) gene reversed the flux from the glycolytic intermediates and led to a higher (-eight-fold) increase in E4P levels [42]. Some non-_Saccharomyces_ yeasts like _Ashyya gossypii_[43], _Kluyveromyces marxianus_[44] and _Candida glycerinogenes_[45] have been investigated for 2-PE production with yields reported that were greater than for _S. cerevisiae._ Similarly, as with _S. cerevisiae_, overexpressing the genes involved in the Ehrlich pathway have led to increased levels in _K. marxianus_[44], but not _A. gossypii_[43]. Media composition, especially adding L-phenylalanine, have shown in many cases to enhance the production of polypropanoids [46; 47]. This implies that the yeast precursor is still a major bottleneck for phenylpropanoloid production.