Systematic Engineering of Saccharomyces cerevisiae Chassis for Efficient Flavonoid-7-O-Disaccharide Biosynthesis

Abstract

Flavonoids are an essential class of secondary metabolites found in plants and possess various nutritional, medicinal, and agricultural properties. However, the poor water solubility of flavonoid aglycones limits their potential applications. To overcome this issue, glycosylation is a promising approach for improving water solubility and bioavailability. In this study, we constructed a flavonoid-7-O-disaccharide biosynthetic pathway with flavonoid aglycones as substrates in Saccharomyces cerevisiae. Subsequently, through metabolic engineering and promoter strategies, we constructed a UDP-rhamnose regeneration system and optimized the UDP-glucose (UDPG) synthetic pathway. The optimized strain produced up to 131.3 mg/L eriocitrin. After this, the chassis cells were applied to other flavonoids, with substrates such as (2S)-naringenin, (2S)-hesperetin, diosmetin, and (2S)eriodictyol, which resulted in the synthesis of 179.9 mg/L naringin, 276.6 mg/L hesperidin, 249.0 mg/L neohesperidin, 30.4 mg/L diosmin, and 100.7 mg/L neoeriocitrin. To the best of our knowledge, this is the first report on the biosynthesis of flavonoid-7-Odisaccharide.

Systematic Engineering of Saccharomyces cerevisiae for the De Novo Biosynthesis of Genistein and Glycosylation Derivatives

“…The biosynthesis of flavonoid glycosides is catalyzed by glucosyltransferases and mediated by UDP sugars [ 22 ]. Genistein was converted to genistin and genistein-8- C -glucoside, catalyzed, respectively, by GmUGT4 and PlUGT43 with the glycosyl donor, UDPG ( Figure 6 a).…”

“…The production of genistin and genistein-8- C -glucoside was further verified using liquid chromatography–mass spectrometry ( Figure 6 c). UDPG production by S. cerevisiae requires the expression of phosphorylase (PGM1/PGM2) and glucose-1-uridyltransferase (UGP1) [ 22 ]. Strain NHGO12-19 was constructed by expressing PGM1/PGM2 alone or in combination with UGP1 in NHGO09-10.…”

“…Optimization of UDPG supply is a commonly used tool in glycosylation pathway construction [ 47 ]. In S. cerevisiae , glucose is first converted to glucose 6-phosphate (G-6-P), then to glucose 1-phosphate (G-1-P) by the action of PGM1/PGM2 , and UDPG is finally catalyzed by UGP1 [ 22 ]. These genes were overexpressed to expand the UDPG pool and overcome the rate-limiting challenges in the glycoside product synthesis pathway ( Figure 6 a).…”

“…Subsequently, 2-hydroxyisoflavone dehydratase (HID) dehydrates 2-hydroxyisoflavone to produce genistein [ 21 ]. Isoflavones are often found in plants as O - or C -glycosides [ 22 ] that are advantageous in terms of solubility, stability, and bioavailability [ 14 ]. Genistein is modified by 7- O -glycosylation to produce genistin, which is more bioavailable and readily absorbed in the intestine [ 23 ].…”

High-Level De Novo Production of (2S)-Eriodictyol in Yarrowia Lipolytica by Metabolic Pathway and NADPH Regeneration Engineering

“…It is broadly used in the pharmaceutical and food industries, serving as an antioxidant, a slimming drug, and an anti-Alzheimer’s disease agent. − Additionally, as a food additive, (2 S )-eriodictyol can be directly applied in food to increase its health benefits . Consuming foods rich in (2 S )-eriodictyol has been confirmed to be beneficial for physical health . Therefore, high-level production of (2 S )-eriodictyol would significantly facilitate the advancements of pharmaceutical and food applications .…”