Glycyrrhetinicacid (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 GlycyrrhetinicAcid3-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 AcGUS3G461C/Q462H/I575K with significantly improved activity (kcat/Km of 11.02-fold) was obtained via computer-aided engineering. Furthermore, the dual-GUS combination strategy was employed for the first timeto 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.

中文翻译：

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通过蛋白质工程和双 GUS 组合策略与来自阿曲霉 CLH-22 的新型 β-葡萄糖醛酸酶进行高效的甘草次酸生物制造


甘草次酸 （GA） 是一种高价值的五环三萜类化合物，具有广泛的应用。然而，GA 的工业生产受到低产量和中间产物甘草次酸 3-O-单β-D-葡萄糖醛酸苷 （GAMG） 积累的阻碍。本研究首先通过转录组学分析从 Aspergillus calidoustus CLH-22 中鉴定出一种新的 β-葡萄糖醛酸酶 （AcGUS），证明了对 GAMG 的底物偏好。随后，通过计算机辅助工程获得了活性显著提高 （kcat/Km 为 11.02 倍） 的突变体 AcGUS3G461C/Q462H/I575K。此外，首次采用双 GUS 组合策略构建用于 GA 生产的工程 Pichia pastoris，具有提高转化效率和降低发酵粘度的多重优势。最后，在系统优化的条件下，采用甘草甜素 （GL） 作为底物，GA 的最终浓度为 48.73 g/L，在 1000 L 发酵罐中的转化率为 97.26 %，代表了迄今为止报道的最佳生物催化性能。本研究为工业 GA 生产提供了新的思路和见解。