# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 2 Yeast as a Recombinant Host for Bioflavour Production
Designer synthetic DNA oligonucleotides allow the combinatorial alteration of pathway genes and, with successive rounds of transformation, gradually remodel the yeast genome toward the production of a metabolite or to embody a specific phenotype. Smaller, directed libraries, only altering the pathway(s) of interest, have been demonstrated with techniques like Versatile Genetic Assembly System (VEGAS). VEGAS uses the yeast's innate preference for homologous recombination to assemble complex pathways, allowing different combinations of the pathway genes to be assembled and subsequently screened for the best production [20]. A new generation of yeasts might allow us to greatly expand yeast strain diversity beyond what has resulted to date with directed breeding and natural selection. The revolutionary synthetic biology initiative known as the Yeast 2.0 project (also known as Sc2.0) was initiated in 2007 [21] to deepen our understanding of the molecular mechanisms that drives this versatile organism. Upon completion, the Sc2.0 strain will be world's first eukaryote with a streamlined chemically-synthesised genome. In addition to the removal of repetitive sequences, the liberation of a codon and the introduction of hundreds of watermark sequences, LoxPsym sequences were introduced at the 5'-ends of all genes considered individually non-essential [22; 23]. These sites allow for inducible homologous recombination downstream of all non-essential genes, mediated by the action of the site-specific Cre-recombinase. Upon activation of the site-specific Cre-recombinase, homologous recombination is promoted between these LoxPsym sites, resulting in rapid gene deletion, duplication or inversion. This process--known as SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution)--allows for the rapid synthetic rearrangement and evolution of the yeast genome [24] (Figure 1). In addition to this novel way of producing large libraries of genomically-divergent yeasts, SCRaMbLE also allows us to produce and explore minimum eukaryotic genomes for the first time. These libraries will be valuable assets in the screening for interesting phenotypes, like aroma compound production and the elucidation of the underlying principles governing these production pathways. Irrespective of the specific yeast strain used, optimisation of the recombinant production of a given protein or metabolite would require the systematic improvement of the properties of the recombinant host using analytical and computational methods to quantify fluxes and their regulation. The following guiding principle questions, regarding global and pathway-specific metabolic engineering, have been proposed previously [26]: (i) can the precursor and/or cofactor supply be increased? ; (ii) can the heterologous expression of non-native genes be different or the expression thereof be improved? ; (iii) can pathways that compete for the same precursors and co-factors be blocked or down-regulated? ; (iv) are transcriptional regulators known and what would be the effect if they are overexpressed? ; and (v) can the enzyme specificity be improved? Most of these questions are directly applicable in improving a yeast's ability to produce aroma compounds. Below we will discuss the work researchers have undertaken in addressing these questions in order to increase the levels of phenylpropanol and terpenoid production in yeast. Figure 1: Depiction of aroma compound pathway optimisation through loxPsym-mediated rearrangement of synthetic chromosomes (SCRaMbLE) in yeast. (**A**) A yeast containing synthetic versions of their respective chromosomes with multiple loxPsym sequences would be subjected to the actions of the loxPsym-specific Cre recombinase. (**B**) The subsequent insertions, duplications, deletions, inversions and other genetic alterations will allow for the generation of an instantly-made library of yeast that have tremendous diversity in their respective genetic backgrounds (**C**) allowing for the screening of yeast with preferred phenotypes.