AUTHOR=Wu Zhigeng , Singh Sanjay Kumar , Lyu Ruiqing , Pattanaik Sitakanta , Wang Ying , Li Yongqing , Yuan Ling , Liu Yongliang 

TITLE=Metabolic engineering to enhance the accumulation of bioactive flavonoids licochalcone A and echinatin in Glycyrrhiza inflata (Licorice) hairy roots

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.932594

DOI=10.3389/fpls.2022.932594

ISSN=1664-462X

ABSTRACT=<p>Echinatin and licochalcone A (LCA) are valuable chalcones preferentially accumulated in roots and rhizomes of licorice (<italic>Glycyrrhiza inflata</italic>). The licorice chalcones (licochalcones) are valued for their anti-inflammatory, antimicrobial, and antioxidant properties and have been widely used in cosmetic, pharmaceutical, and food industries. However, echinatin and LCA are accumulated in low quantities, and the biosynthesis and regulation of licochalcones have not been fully elucidated. In this study, we explored the potential of a R2R3-MYB transcription factor (TF) <italic>AtMYB12</italic>, a known regulator of flavonoid biosynthesis in <italic>Arabidopsis</italic>, for metabolic engineering of the bioactive flavonoids in <italic>G. inflata</italic> hairy roots. Overexpression of <italic>AtMYB12</italic> in the hairy roots greatly enhanced the production of total flavonoids (threefold), echinatin (twofold), and LCA (fivefold). RNA-seq analysis of <italic>AtMYB12</italic>-overexpressing hairy roots revealed that expression of phenylpropanoid/flavonoid pathway genes, such as <italic>phenylalanine ammonia-lyase</italic> (<italic>PAL</italic>), <italic>chalcone synthase</italic> (<italic>CHS</italic>), and <italic>flavanone 3’-hydroxylase</italic> (<italic>F3’H</italic>), is significantly induced compared to the control. Transient promoter activity assay indicated that AtMYB12 activates the <italic>GiCHS1</italic> promoter in plant cells, and mutation to the MYB-binding motif in the <italic>GiCHS1</italic> promoter abolished activation. In addition, transcriptomic analysis revealed that <italic>AtMYB12</italic> overexpression reprograms carbohydrate metabolism likely to increase carbon flux into flavonoid biosynthesis. Further, AtMYB12 activated the biotic defense pathways possibly by activating the salicylic acid and jasmonic acid signaling, as well as by upregulating WRKY TFs. The transcriptome of <italic>AtMYB12</italic>-overexpressing hairy roots serves as a valuable source in the identification of potential candidate genes involved in LCA biosynthesis. Taken together, our findings suggest that <italic>AtMYB12</italic> is an effective gene for metabolic engineering of valuable bioactive flavonoids in plants.</p>