Metabolic Engineering to Produce Rosmarinic Acid in Baker´s Yeast Saccharomyces Cereviciae

Rosmarinic acid (RA) is a secondary plant metabolite with antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Currently the rosmarinic acid is obtained by extraction from plants. Microbial production of RA is an attractive alternative, which can save time and cut the production costs. In this study we aimed to engineer the yeast Saccharomyces cereviciae to produce RA from a cheap carbon source, e.g., glucose. First, we reconstructed two variants of biosynthetic pathway from p-coumaric acid to RA, a plant-like pathway and a chimeric microbial-plant pathway, and obtained 20 ± 4 and 18 ± 3 µg·L-1 of RA respectively, when culturing the cells with p-coumaric acid. Second, we overexpressed tyrosine ammonia lyase Flavobacterium johnsoniae to obtain RA directly from glucose without supplementation of precursor. Both the plant-like pathway and the chimeric pathway resulted in similar titers of ~10 µg·L-1, when culturing the cells on minimal medium with glucose as the sole carbon source. Furthermore we engineered the strains to increase the supply of precursors, p-coumaric acid and 4-hydroxyphenyllactic acid, by overexpressing ARO4fbr & ARO7fbr and knocking out PDC5 and ARO10, leading to an increase of 2.8-fold of p-coumaric acid and 3.1-fold in 4-hydroxyphenyllactic acid compared to the non-engineered strains and with no significant effect on RA production. Subsequently, the copy number of rosmarinic acid synthase-encoding gene was increased using either a multiple integration into the genome or expression from a 2µ-vector to result in 3.3 and 6.6-fold increase of RA for plant and chimeric pathways respectively, compared to the parental strains. Finally, cultivating the best producing strain in feed-in-time medium resulted in the final concentration of 256±25 µg·L-1 of RA. To our knowledge this is the first time rosmarinic acid was produced in S. cerevisiae.