De Novo Production of Hydroxytyrosol by Saccharomyces cerevisiae–Escherichia coli Coculture Engineering

Hydroxytyrosol is a valuable plant-derived phenolic compound with excellent pharmacological activities for application in the food and health care industries. Microbial biosynthesis provides a promising approach for sustainable production of hydroxytyrosol via metabolic engineering. However, its efficient production is limited by the machinery and resources available in the commonly used individual microbial platform, for example, Escherichia coli, Saccharomyces cerevisiae. In this study, a S. cerevisiae-E. coli coculture system was designed for de novo biosynthesis of hydroxytyrosol by taking advantage of their inherent metabolic properties, whereby S. cerevisiae was engineered for de novo production of tyrosol based on an endogenous Ehrlich pathway, and E. coli was dedicated to converting tyrosol to hydroxytyrosol by use of native hydroxyphenylacetate 3-monooxygenase (EcHpaBC). To enhance hydroxytyrosol production, intra- and intermodule engineering was employed in this microbial consortium: (I) in the upstream S. cerevisiae strain, multipath regulations combining with a glucose-sensitive GAL regulation system were engineered to enhance the precursor supply, resulting in significant increase of tyrosol production (from 17.60 mg/L to 461.07 mg/L); (II) Echpabc was overexpressed in the downstream E. coli strain, improving the conversion rate of tyrosol to hydroxytyrosol from 0.03% to 86.02%; (III) and last, intermodule engineering with this coculture system was performed by optimization of the initial inoculation ratio of each population and fermentation conditions, achieving 435.32 mg/L of hydroxytyrosol. This S. cerevisiae-E. coli coculture strategy provides a new opportunity for de novo production of hydroxytyrosol from inexpensive feedstock.