De Novo Production of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide in Saccharomyces cerevisiae

Abstract

Glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) is a rare compound in licorice and its short supply limits the wide applications in the pharmaceutical, cosmetic, and food industries. In this study, de novo biosynthesis of GAMG was achieved in engineered Saccharomyces cerevisiae strains based on the CRISPR/Cas9 genome editing technology. The introduction of GAMG biosynthetic pathway resulted in the construction of a GAMG-producing yeast strain for the first time. Through optimizing the biosynthetic pathway, improving the folding and catalysis microenvironment for cytochrome P450 enzymes (CYPs), enhancing the supply of UDP-glucuronic acid (UDP-GlcA), preventing product degradation, and optimizing the fermentation conditions, the production of GAMG was increased from 0.02 μg/L to 92.00 μg/L in shake flasks (4,200-fold), and the conversion rate of glycyrrhetic acid (GA) to GAMG was higher than 56%. The engineered yeast strains provide an alternative approach for the production of glycosylated triterpenoids.

Production of Plant Sesquiterpene Lactone Parthenolide in the Yeast Cell Factory

“…Glucose-6-phosphate dehydrogenase (ZWF1) and NADH kinase (POS5) are effective NADPH-supplying sources in yeast (Figure A). , Specifically, the POS5 lacking the coding sequence for the first 17 amino acid residues, designated POS5Δ17, can efficiently regenerate NADPH from NADH during the conversion process from ATP to ADP. , ZWF1 responsible for the reaction from glucose-6-phosphate to 6-phosphogluconolactone can convert NADP + to NADPH . Huang et al increased NADPH by overexpressing GAPN (glyceraldehyde-3-phosphate dehydrogenase) and ZWF1 , and the production of glycyrrhetic acid and glycyrrhetic acid 3-O-mono-β- d -glucuronide was 1.4- and 1.7-fold higher than that of the control, respectively . Thus, POS5Δ17 and ZWF1 were chosen for investigation.…”

“…36 Huang et al increased NADPH by overexpressing GAPN (glyceraldehyde-3-phosphate dehydrogenase) and ZWF1, and the production of glycyrrhetic acid and glycyrrhetic acid 3-O-mono-β-D-glucuronide was 1.4-and 1.7-fold higher than that of the control, respectively. 40 Thus, POS5Δ17 and ZWF1 were chosen for investigation. ZWF1 and POS5Δ17 were individually overexpressed under the constitutive TDH3 promoter by integrating them in the genome of ySYT017 at the gal 1/7/10 locus, respectively, obtaining the strains ySYT055 and ySYT056 (Figure 3B).…”

Specific Glycosylation System Enables the High-Level and Sustainable Production of Glycyrrhetinic Acid 3-O-monoglucuronide and Glycyrrhizin in Engineered Saccharomyces cerevisiae

“…Glycosyltransferase GuGT14 was utilized to catalyze the glucuronidation of GA to GAMG, obtaining a GAMG titer of 92 μg/L by optimizing the catalytic microenvironment and culture conditions and strengthening precursor supply. 13 In addition to plant-derived glycosyltransferases, UGT1A1 from Homo sapiens (HsUGT1A1) was used to catalyze consecutive two-step glucuronidation to convert GA into GL, producing 5.98 and 2.31 mg/L GL and GAMG in S. cerevisiae, respectively, which are the highest levels reported to date. 12 However, the activity of HsUGT1A1 was considerably low, making it difficult to further improve the titers of GAMG and GL.…”

“…The glycosidic bonds in GAMG and GL are both in the β configuration. The introduction of GlcA group not only improves the water solubility of GA but also turns GA into natural, low-calorie sweeteners, with GAMG and GL being 941 and 170 times sweeter than sucrose, respectively. , Currently, most glycosides are glucosyl modified, and glucuronide-modified glycosides are less studied and of great importance.…”

“…Subsequently, our group achieved the complete heterologous synthesis of GA from sugar in Saccharomyces cerevisiae with a titer of 36.4 mg/L. , In the past 2 years, there were some efforts to realize the heterologous de novo biosynthesis of GAMG and GL. Glycosyltransferase GuGT14 was utilized to catalyze the glucuronidation of GA to GAMG, obtaining a GAMG titer of 92 μg/L by optimizing the catalytic microenvironment and culture conditions and strengthening precursor supply . In addition to plant-derived glycosyltransferases, UGT1A1 from Homo sapiens (HsUGT1A1) was used to catalyze consecutive two-step glucuronidation to convert GA into GL, producing 5.98 and 2.31 mg/L GL and GAMG in S.…”