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.

Keywords: CRISPR/Cas9; Saccharomyces cerevisiae; cytochrome P450 enzymes (CYPs); glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG); metabolic engineering.

FIGURE 1

The biosynthetic pathway of GAMG. (A) Chemical structure of GA, GAMG, and GL. (B) Metabolic pathway for GAMG synthesis in S. cerevisiae. GAMG was synthesized from GA and UDP-GlcA by UGTs. The whole GAMG biosynthetic pathway was divided into three functional modules: the GA Module (Module I), the UDP-GlcA Module (Module II), and the glycosylation Module (Module III). IPP: isopentenyl pyrophosphate; FPP: farnesyl diphosphate; GA: glycyrrhetinic acid. GAMG: glycyrrhetic acid 3-O-mono-β-d-glucuronide; GL: glycyrrhizin; UDP-Glc: uridine diphosphate-glucose; UDP-GlcA: uridine diphosphate glucuronic acid.

FIGURE 2

Optimization of the GA module (module I) to improve GAMG production. (A) Optimization of GA biosynthetic pathway for improved GAMG production. Endogenous genes overexpressed in the parent strain BY4741-C-04 were shown in green, endogenous genes overexpressed in the present study were shown in blue, and heterologous genes overexpressed to synthesize GA were shown in red. ZWF1 and GAPN were overexpressed to enhance NADPH supply and OPI1 was disrupted to provide more space for the folding of CYPs via ER membrane expansion. (B) Comparison of GA and GAMG production in engineered S. cerevisiae strains. ERG10: acetyl-CoA acetyltransferase; ERG13: hydroxymethylglytaryl-CoA synthase; tHMG1: truncated-3-methylglutaryl-CoA reductase; ERG12: mevalonate kinase; ERG8: phosphomevalonate knase; ERG19: diphosphomevalonate decarboxylase; IDI1: dimethylallyl diphosphate isomerase; ERG20: farnesyl pyrophosphate synthase; ERG9: squalene synthase; ERG1: squalene epoxidase; βAS: β-amyrin synthase; GuCPR1: cytochrome P450 reductase from Glycyrrhiza uralensis; UNI25647: β-amyrin 11-oxidase; mutCYP72A63: the T388S/W205R mutant of 11-oxo-β-aymrin 30-oxidase; GAPN: NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans; OPI1: OverProducer of Inositol; ZWF1: glucose-6-phosphate dehydrogenase; ER: endoplasmic reticulum. Error bars represented SD of biological triplicates.

FIGURE 3

Optimization of the UDP-GlcA module (module II) for improving GAMG production, including the prevention of GAMG degradation and enhancement of UDP-GlcA supply. (A) UDP-GlcA biosynthetic pathway and its involvement in GA glycosylation. PGM1, PGM2, UGP1, and AtUDH were overexpressed to enhance UDP-GlcA supply. EGH1 was deleted to prevent GAMG degradation. (B) Comparison of GA and GAMG production in engineered S. cerevisiae strains. PGM1: phosphoglucomutase 1; PGM2: phosphoglucomutase 2; UGP1: UDP-glucose pyrophosphorylase; AtUDH: UDP-glucose dehydrogenase from A. thaliana; GuUGT73F15: UDP-glucuronyltransferase. Error bars represented SD of biological triplicates.

FIGURE 4

Optimization of carbon sources for enhanced GAMG production in yeast strain GA15. GAMG and GA fermentation were carried out in YP medium supplemented with 2% galactose, 2% glucose, or different concentration of glycerol. Error bars represent SD of biological triplicates. Gal: galactose; Glu: glucose; Gly: glycerol.