# Heterologous Production of Flavour and Aroma Compounds in _Saccharomyces cerevisiae_
## 2 Heterologous Compounds Derived from Intermediates of the Shikimate Pathway  
**Vanillin**, (4-hydroxy-3-methoxybenzaldehyde), is a plant secondary metabolite commonly extracted from the seed pods of the vanilla orchids _Vanilla planifolia_, _Vanilla talitensis_, or _Vanilla pompona_. It is one of the most commonly used food and beverage flavouring additives, with over 16,000 tonnes of vanillin consumed worldwide each year [6]. However, the slow growth of the vanilla orchid combined with the relatively low content of vanillin in the mature seed pods, means that only a minor fraction (\(\sim\)0.25%) of the total production of this compound is actually derived from vanilla orchids [7]. Market demand for this flavouring agent is therefore mostly fulfilled through chemical synthesis from lignin and/or fossil hydrocarbons, processes that are regarded as environmentally unfriendly and not sustainable. Alternative means of non-synthetic vanillin production are therefore highly desirable [8]. Heterologous biosynthetic production of vanillin by _S. cerevisiae_ is one of the most widely recognized examples of applying a synthetic biology system for the industrial production of a highvalue compound via a microbial cell factory. The metabolic pathway that allows yeast to produce vanillin was first developed nearly a decade ago, requiring the expression of four heterologous enzymes; 3-dehydrooshikimate dehydatase (3 DSD) from the dung mold _Podospora pauciseta_, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the _Nocardiia_ genus, an \(O\)-methyltransferase from _Homo sapiens_ (_Hs_OMT), and a phosphopantetheinyl transferase from _Corynebacterium glutamic_ (EntD); the latter enzyme was required for activation of ACAR via phosphopantetheinylation ([PERSON] _et al._, 2016) (Figure 1). Introduction of these foreign genes provided _S. cerevisiae_ with the ability to convert the endogenous metabolic precursor 3-dehydrooshikimate (3-DHS), an intermediate of the shikimate pathway for aromatic amino acid biosynthesis, into vanillin at concentrations approaching 45 mg/L. However, these initial attempts at optimizing the production of vanillin were complicated by the reported toxicity of vanillin against many microorganisms, including yeast, where concentrations exceeding 50 mg/L can significantly inhibit growth ([PERSON] _et al._, 2016), thereby imposing a ceiling on maximum vanillin production levels. The problem of vanillin toxicity was solved via the heterologous expression of a glycosyltransferase (UGT) from _Arabidopsis thaliana_, which converted vanillin into its less toxic and more soluble form, vanillin-\(b\)-d-glucoside (VG). This strategy, along with flux engineering of the native yeast metabolism, involving removal of the pyruvate decarboxylase (_PDC1_) and glutamate dehydrogenase (_GDH1_) genes, as well as overexpression of glutamate dehydrogenase (_GDH2_), resulted in a vanillin production level of 500 mg/L. However, despite this significant increase, these levels were still far below the theoretical yields predicted through metabolic modelling analysis ([PERSON] _et al._, 2016). To remove further metabolic bottlenecks, two potentially rate limiting heterologous steps in the vanillin synthesis pathway were addressed by additional gene overexpression; ACAR, which converts protocatechic acid (PAC) to protocatechuic aldehyde (PAL), and _Hs_OMT which catalyses the conversion of (PAL) to vanillin (Figure 1). While overexpression of ACAR had no effect on final VG production, a 30% improvement was observed in the _Hs_OMT overexpressing strain ([PERSON] _et al._, 2016). In an alternative study, [PERSON] and colleagues ([PERSON] _et al._, 2016) investigated the impact of different yeast strains on the VG production. By reconstituting heterologous VG biosynthesis pathway in two commonly used yeast strains, _S. cerevisiae_ CEN.PK and S288c, it was observed that the S288c background was able to produce up to a 10-fold greater concentration of VG under the same fermentation conditions.