AUTHOR=Su Xinglong , Meng Fei , Liu Yingying , Jiang Weimin , Wang Zhaojian , Wu Liping , Guo Xiaohu , Yao Xiaoyan , Wu Jing , Sun Zongping , Zha Liangping , Gui Shuangying , Peng Daiyin , Xing Shihai TITLE=Molecular Cloning and Functional Characterization of a β-Glucosidase Gene to Produce Platycodin D in Platycodon grandiflorus JOURNAL=Frontiers in Plant Science VOLUME=13 YEAR=2022 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.955628 DOI=10.3389/fpls.2022.955628 ISSN=1664-462X ABSTRACT=

Platycodin D (PD) is a deglycosylated triterpene saponin with much higher pharmacological activity than glycosylated platycoside E (PE). Extensive studies in vitro showed that the transformation of platycoside E to platycodin D can be achieved using β-glucosidase extracted from several bacteria. However, whether similar enzymes in Platycodon grandiflorus could convert platycoside E to platycodin D, as well as the molecular mechanism underlying the deglycosylation process of platycodon E, remain unclear. Here, we identified a β-glucosidase in P. grandiflorus from our previous RNA-seq analysis, with a full-length cDNA of 1,488 bp encoding 495 amino acids. Bioinformatics and phylogenetic analyses showed that β-glucosidases in P. grandiflorus have high homology with other plant β-glucosidases. Subcellular localization showed that there is no subcellular preference for its encoding gene. β-glucosidase was successfully expressed as 6 × His-tagged fusion protein in Escherichia coli BL21 (DE3). Western blot analysis yielded a recombinant protein of approximately 68 kDa. In vitro enzymatic reactions determined that β-glucosidase was functional and could convert PE to PD. RT-qPCR analysis showed that the expression level of β-glucosidase was higher at night than during the day, with the highest expression level between 9:00 and 12:00 at night. Analysis of the promoter sequence showed many light-responsive cis-acting elements, suggesting that the light might regulate the gene. The results will contribute to the further study of the biosynthesis and metabolism regulation of triterpenoid saponins in P. grandiflorus.