Efficient glycyrrhetinic acid biomanufacturing through protein engineering and dual-GUS combination strategy with novel β-glucuronidase from Aspergillus calidoustus CLH-22

Glycyrrhetinic acid (GA) is a high-value pentacyclic triterpenoid with broad applications. However, the industrial production of GA is hindered by low yield and the accumulation of the intermediate product Glycyrrhetinic Acid 3-O-Mono-β-D-Glucuronide (GAMG). This study first identified a novel β-glucuronidase (AcGUS) from Aspergillus calidoustus CLH-22 through transcriptomic analysis, demonstrating a substrate preference for GAMG. Subsequently, mutant AcGUS3^{G461C/Q462H/I575K} with significantly improved activity (k_cat/K_m of 11.02-fold) was obtained via computer-aided engineering. Furthermore, the dual-GUS combination strategy was employed for the first time to construct engineered Pichia pastoris for GA production, offering multiple advantages of enhanced conversion efficiency and reduced fermentation viscosity. Finally, under systematically optimized conditions and employing Glycyrrhizin (GL) as the substrate, the final concentration of GA was 48.73 g/L with a conversion of 97.26 % in a 1000-L fermenter, representing the optimal biocatalytic performance reported to date. This study provides new ideas and insights for industrial GA production.