# The Small of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast
## 2 Yeast as a Recombinant Host for Bioflavour Production  
Various yeasts--with _S. cerevisiae_ being the model organism--have long been harnessed for the expression of recombinant genes to enhance endogenous aroma-active metabolites of the host cells or to produce novel recombinant compounds. The initial reasons why researchers opted for _S. cerevisiae_ remains true, that is, this yeast species is by far the best-studied unicellular eukaryote with the genomes of several of its strains fully sequenced [12]; it is a non-pathogen that enjoys GRAS (generally recognised as safe) status; and it is amenable to genetic manipulation with a wide range of genetic tools available to alter the genetic make-up of the yeast. _S. cerevisiae_ also possesses an efficient homologous recombination machinery, which greatly assists stable integration of genetic elements. This yeast is also the most robust fermenter and laboratory-scale processes can be scaled up to industrial-level set-ups with relative ease. Some of the abovementioned attributes also hold true for _E. coli._ However, as a prokaryote, this bacterium lacks a sophisticated protein-folding mechanism. This often leads to the recombinant proteins being insoluble and most likely non-functional and that might require additional recovery steps for refolding of the protein of interest. _S. cerevisiae_ is, however, by no means a perfect host; for example, it is not a prolific biomass producer and the way secreted proteins are glycosylated sometimes lead to pronounced reduction in bioactivity. There are also reports of recombinant genes that cannot be successfully expressed for unknown reasons. Regardless of the whether _S. cerevisiae_ turns out to be appropriate as a host to produce a particular recombinant product, it remains the best starting point to move onwards to other organisms. A prudent strategy is to examine the expression levels in multiple yeast hosts and to compare titres of a protein (or metabolite) of interest. Often the methylotrophic yeast _Pichia pastoris_ (now reclassified as _Komagataella phiffi_) and _Hansenula polymorpha_ (now reclassified as _Ogataea polymorpha_) have shown superior protein and/or metabolite production capabilities owing to their unusually high biomass production [13]. Many other yeast species with their own special attributes can (and have) been utilised as a recombinant host with varying outcomes. Examples of such yeasts include _Kluyveromyces lactis_, _Yarrowia lipolytica_ and _Schizosaccharomyces pombe_. The usefulness of non-_Saccharomyces_ yeasts in the biotransformations of certain substrates into aroma compounds with whole-cell or resting cell systems are well-documented [14]. This has been a popular way of producing aroma compounds as it can allow for the assembly of regio- and stereoselective compounds under mild and mostly solvent-free conditions. Identification of natural variation within a yeast strains and species has undeniably created a valuable source of flavour-active strains [15]. The underlying molecular determinants for a particular phenotype has been elucidated through the advances in 'omics' capability. Effective mining of genes and alternative alleles responsible for a desired phenotype have become common practice, with access to comprehensive conventional yeast libraries based on mutagenesis, breeding [16], single gene deletions [17] and overexpression [18]. Yeast libraries have become more sophisticated and, in many cases, combine the genomic variation generation with a selection for the particular characteristic of interest. This includes biosensor-enabled directed evolution (discussed in later section below), rapid genome-wide editing (YOGE) or the complete reconstruction of pathways (VEGAS) and genomes (Yeast 2.0). Yeast Oligo-mediated Genome Engineering (YOGE) enables rapid genome engineering by introducing allele variation by sequential oligonucleotide recombination [19].