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.
Highlights
Licorice is a popular and traditional Chinese medicinal plant and is listed in the “Medicine Food Homology” published by the Ministry of Health (China)
The biosynthesis of Glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) in yeast mainly consisted of pathways from acetyl-CoA to glycyrrhetic acid (GA) and from sugar to UDP-glucuronic acid, followed by O-glycosylation by the glycosyltransferase (Figure 1B)
The GAMG biosynthetic pathways were divided into three functional modules, the GA module (Module I), the UDP-glucuronic acid (GlcA) module (Module II), and the glycosylation module (Module III)
Summary
Licorice is a popular and traditional Chinese medicinal plant and is listed in the “Medicine Food Homology” published by the Ministry of Health (China). Its roots contain plenty of strong bioactive compounds, such as triterpenoids and flavonoids (Gao et al, 2009). Two of the predominant bioactive triterpenoids are glycyrrhizin (GL) and glycyrrhetinic acid (GA), which show many valuable pharmacological properties including anti-inflammatory, anti-cancer, anti-allergic, and anti-virus effects (Wang et al, 2015), and are widely used as natural sweeteners in candies and canned foods. In addition to the major triterpenoids, there are several minor compounds with high-value biological activities in licorice, in particular glycyrrhetinic acid monoglucuronide (3-O-β-D-glucuronopyranosyl glycyrrhetinic acid, GAMG). GAMG has been shown to have similar pharmacological effects as GA and GL (Guo et al, 2018). GAMG can be used as a natural innovative sweetener with health effects in food industry, because it is 5- and 941-times sweeter than GL and sucrose, respectively. Considering the great importance and the expanded market of GAMG as a valuable
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