De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae

Hydroxytyrosol is an olive-derived phenolic compound of increasing commercial interest due to its health-promoting properties. In this study, a high-yield hydroxytyrosol-producing Saccharomyces cerevisiae cell factory was established via a comprehensive metabolic engineering scheme. First, de novo biosynthetic pathway of hydroxytyrosol was constructed in yeast by gene screening and overexpression of different phenol hydroxylases, among which paHD (from Pseudomonas aeruginosa ) displayed the best catalytic performance. Next, hydroxytyrosol precursor supply was enhanced via a multimodular engineering approach: elimination of tyrosine feedback inhibition through genomic integration of aro4 K229L and aro7 G141S, construction of an aromatic aldehyde synthase (AAS)-based tyrosine metabolic pathway, and redistribution of metabolic flux between glycolytic pathway and pentose phosphate pathway (PPP) by introducing the exogenous gene Bbxfpk opt . As a result, the titer of hydroxytyrosol was improved by 6.88-fold. Finally, a glucose-responsive dynamic regulation system based on GAL80 deletion was implemented, resulting in the final hydroxytyrosol yields of 308.65 mg/L and 167.98 mg/g cell mass, the highest known from de novo production in S. cerevisiae to date.