Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis

Abstract

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.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[134] The usage of feedback resistant DAHP synthase was also effective in yeast for enhanced p-coumaric acid production. [136] The authors overexpressed various feedback resistant DAHP synthases (ARO4 K229L from S. cerevisiae, aroF and two mutant variants of aroG from E. coli) and chorismate mutases (ARO7 G141S from S. cerevisiae, a chorismate mutase from C. guilliermonii, and tyrA from the E. coli). Among the various combinations, ARO4 K229L /ARO7 G141S was the best pair when they were overexpressed with TAL from Flavobacterium johnsoniaeu and aroL encoding shikimate kinase II from E. coli.…”

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

“…Increasing chorismate levels is more complex and needs to be carefully balanced with downstream product formation due to the high instability of chorismate (Winter et al, 2014). Nevertheless, overexpression of aroL has been shown to significantly increase flux through shikimate pathway (Rodriguez et al, 2015). Preliminary results (unpublished data) show that this is also valid for the production of pHBA.…”

“…Using metabolic engineering these capabilities are already being exploited to create new organisms that use abundant and renewable feedstocks to efficiently produce an expanding spectrum of valuable chemicals (Jang et al, 2012. In particular aromatics have great potential for bio-based production as the shikimate pathway gives rise to a wealth of aromatics and derived compounds, with diverse applications in different industries, including the chemical one (Hansen et al, 2009, Curran et al, 2013, Krömer et al, 2013, Jendresen et al, 2015, Rodriguez et al, 2015, Williams et al, 2015. The shikimate pathway intermediate para-aminobenzoic acid (pABA) is one of these aromatics with versatile applicability.…”

“…genes were deleted and the feedback inhibition resistant ARO4 K229L gene was introduced, in order to overcome product inhibition and increase flux to the shikimate pathway (Curran et al, 2013, Rodriguez et al, 2015, Williams et al, 2015. In the resulting genetically modified base strain "PABA0" (cf.…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…of p-coumaric acid as the end-product in S. cerevisiae, through optimization of native aromatic amino acid biosynthesis [54]. The competing pathways were eliminated while enhancing production pathways by the expression of feedback resistant enzymes in combination with gene deletions and overexpression of analogue enzymes from E. coli.…”