Engineering of Saccharomyces cerevisiae for the production of natural & synthetic products

Biochemical pathway engineering represents an economical and environmentally responsible option for the synthesis of valuable compounds. In this study, an engineered strain of Saccharomyces cerevisiae was used to produce some of these molecules by expressing two plant enzymes, 4-coumaroyl ligase (4CL5) and N-Hydroxycinnamoyl/benzoyl transferase (HCBT). Engineered yeast strains expressing these enzymes were monitored for substrate depletion and product accumulation via liquid chromatography and mass spectrometry (LC/MS). With the aim of reducing production costs, potential toxicity of supplied substrates, and increasing yields, a bacterial Tyrosine Ammonia-Lyase (TAL) was also co-expressed in yeast to enable the conversion of tyrosine (yeast endogenous substrate) into coumaric acid, an important precursor of our target products. We successfully demonstrated that TAL converts endogenous and exogenous tyrosine into coumaric acid when expressed in yeast, and the production of coumaroyl-A thioesters that are further coupled to different acceptors when co-expressed with 4CL5 and HCBT enzymes. Expression of this pathway in yeast provided a route for in vivo synthesis from glucose of the coumarate-ester avenanthramide-D among other compounds. Additionally, an expression system was built for the production of phenylacetate using phenylacetaldehyde dehydrogenase (PADH). The conversion of phenylacetaldehyde into phenylacetate (another relevant compound precursor) via PADH is known to occur in the upper pathway of styrene degradation in Pseudomonas putida (S12).