High-level sustainable production of complex phenylethanoid glycosides from glucose through engineered yeast cell factories.

Complex phenylethanoid glycosides (PhGs), such as verbascoside and echinacoside, comprise a vital family of natural products with renowned nutraceutical and pharmaceutical significance. Despite the high demand for these compounds across various industries, traditional plant extraction methods yield insufficient quantities, highlighting the need for alternative production methods. Therefore, this paper reports the successful engineering of Saccharomyces cerevisiae cell factories for the efficient production of complex PhGs from glucose. First, key pathway enzymes with enhanced catalytic activities in yeast were primarily screened from various verbascoside-producing plants. Second, intermediate osmanthuside B was produced with a titer of 21.5 ± 1.5 mg/L from glucose by overexpressing several enzymes, including glucosyltransferase RrUGT33 from Rhdiola rosea, acyltransferase SiAT, and 1,3-rhamnosyltransferase SiRT from Sesamum indicum, UDP-L-rhamnose synthase AtRHM2, and 4-coumarate: coenzyme A ligase At4CL1 from Arabidopsis thaliana in a p-coumaric acid-overproducing S. cerevisiae strain. Third, the production of osmanthuside B was further enhanced by increasing the copy number of SiAT and AtRHM2 in genome and diverting L-tyrosine into tyrosol biosynthesis by introducing an aromatic aldehyde synthase PcAAS from Petroselinum crispum with a titer of 320.6 ± 59.3 mg/L. Fourth, the biosynthesis of verbascoside was accomplished by integrating genes CYP98A20 and AtCPR1 into the chromosomes of the osmanthuside B-producing strain, the titer reached 184.7 ± 5.7 mg/L. Furthermore, the overexpression of the glucose-6-phosphate dehydrogenase (ZWF1) led to significantly enhanced verbascoside production to 230.6 ± 11.8 mg/L. The strains were further engineered to produce echinacoside with a titer of 184.2 ± 11.2 mg/L. Finally, the fed-batch fermentation in a 5-L bioreactor yielded 4497.9 ± 285.2 mg/L of verbascoside or 3617.4 ± 117.4 mg/L of echinacoside. This work provides a crucial foundation for the green, industrial, and sustainable production of verbascoside and echinacoside and sets an initial point for the microbial production of other complex PhG derivatives.