Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-knockout variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-knockout and CYP88D6-overexpression. This strategy resulted in a three-fold increase (~1.4mg/g) in glycyrrhizin accumulation in double-knockout/CYP88D6-overexpression hairy roots, on average, compared with that of double-knockout hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

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基于基因组编辑三萜生物合成途径代谢重定向的甘草毛状根中的甘草酸生产

甘草甜素是三萜皂苷的一种，是药用植物甘草（乌拉尔甘草、光果甘草和膨胀甘草）根部含有的主要活性成分，在世界范围内具有多种用途，例如草药和甜味剂。对甘草的需求不断增长威胁着野生资源，因此需要一种可持续的甘草甜素供应方法。为了建立一种不依赖野生植物的替代甘草酸供应方法，我们尝试使用毛状根培养法生产甘草酸。我们试图通过使用基于 CRISPR/Cas9 的基因编辑阻断竞争途径来促进甘草甜素的生产。生成了CYP93E3 CYP72A566双敲除突变体和CYP93E3 CYP72A566 CYP716A179 LUS1四敲除突变体，并且在两种类型的毛状根中都证实了大量的甘草甜素积累。此外，我们评估了通过同时 CYP93E3 CYP72A566 双敲除和 CYP88D6 过表达进一步促进甘草甜素生产的潜力。与双敲除毛状根相比，该策略导致双敲除/CYP88D6过表达毛状根中甘草甜素积累平均增加三倍（~1.4mg/g）。这些发现表明，阻断竞争途径和生物合成基因的过度表达相结合对于提高乌拉尔甘草毛状根中甘草甜素的产量非常重要。我们的研究结果为利用毛状根培养可持续生产甘草甜素奠定了基础。鉴于基因组编辑技术在毛状根中的广泛应用，结合基因敲除和过度表达可以广泛应用于生产各种植物根中含有的有价值的物质。