Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93 ± 0.26 g L(-1) was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4(K229L), chorismate mutase ARO7(G141S) and E. coli shikimate kinase II (aroL) in Δpdc5Δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides.

Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73 ± 0.05 mg L(-1) resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85 ± 0.31 mg L(-1) resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39 ± 0.03 mg L(-1). Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57 ± 7.00 mg L(-1). Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41 mg L(-1), respectively.

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