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(G)(141S), 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(op)(t). 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.