Researchers reveal the formation mechanism of triterpene saponin diversity in the Pentagram family

On April 25, 2022, the team of Professor Li Yuhua of Northeast Forestry University and the team of Professor Shen Hailong published a research paper entitled “Deletion and tandem duplications of biosynthetic genes drive the diversity of triterpenoids in Aralia elata” at Nature Communications. The study reported for the first time the accumulation of rich and diverse pentacyclic triterpene saponins in the pentagram family longataca sylvestris due to the deletion of the damene glycol synthase gene and the tandem repetition of specific modification enzymes at triterpenoid skeleton sites.

Dragon tooth tree (Aralia elata) is a perennial shrub of the genus Aralia elata, which is an important food and drug homologous plant mainly produced in the forest area of northeast China, and is also known as the “king of mountain wild vegetables”, with anti-cancer, anti-inflammatory, antioxidant and treatment of cardiovascular and cerebrovascular diseases, triterpene saponin is its main active ingredient. Although the genus is closely related to the genus Ginseng, the “king of the herbs” (the same Aralia-Panax subpopulation), there are significant differences in the structural type and abundance of triterpene saponins between the two. Ginseng can synthesize a large number of damane-type tetracyclic triterpenes saponins, and only 5 kinds of oleanolane-type pentacyclic triterpene saponins are accumulated, while the damane-type tetracyclic triterpene saponins are not synthesized in the dragon’s tooth tree, but more than 100 kinds of oleracene-type pentacyclic triterpene saponins are accumulated. The structure of triterpene saponins of the Pentaca family plants is significantly different, and the pharmacological activity is also very different, and the biosynthesis and structural diversity mechanism of triterpenes saponins has attracted wide attention at home and abroad.

In this study, the high-quality dragon tooth sycamore genome atlas was constructed by using PacBio three-generation sequencing technology and Hi-C technology. It was found that the species differentiation of the genus was found about 12.3 million years ago with the ancestors of the genus Ginseng, and the synonymous replacement rate (KS) and genomic collinear analysis revealed whole genome replication (WGD) events shared by plants of the pentagram family such as ginseng, panax notoginseng, basswood and eleuthero, and no species-specific WGD events were found in the sassafras. Comparing ginseng, panax notoginseng and other genomes, it was found that the exon region of the damene glycol synthase (DDS) gene that catalyzed the synthesis of 2,3 epoxy squalene into damarene glycol in the argyle occurred in a large number of deletions, and only the amino acid residues encoded by 5 exons were retained, and it was speculated that the deletion of the DDS gene may lead to the inability of the sassafras to synthesize damane-type tetracyclic triterpene saponins. Through transgenic function complementary experiments, the PgDDS gene of ginseng was overexpressed in the callus cells of the sassafras, and the ability of transgenic aqueous sycamore cells to synthesize damalane-type tetracyclic triterpene saponins was restored. These results reveal for the first time the absence of exons in the genome of Dracaena and the key enzyme gene responsible for catalyzing marane-type saponins , resulting in the inability of Dragon’s Tooth Toys to synthesize damalene-type saponins ( Figure 1 ) .

Figure 1 Deletion of the synthase gene of the dragon tooth tree damamenediol affects the synthesis of tetracyclic triterpenes saponins

The study also found that after differentiation from ginseng plants, a large number of tandem replicates occurred in the genome of the key modified enzyme genes P450, CSLM (encoding the M subfamily of cellulose synthase), UGT, etc. involved in triterpene saponin biosynthesis (Figure 2). The functions of 8 key enzyme genes in four tandem repeat regions were identified by using the expression system of Escherichia coli and yeast and the verification of catalytic functions in vivo and in vitro (Figure 3). Finally, by synthetic biology, Saccharomyces cerevisiae was used as a chassis cell, and up to 13 different structures of basso saponins were synthesized from scratch using a combination of different types of enzymes.

Fig. 2 Triterpenoidal modified enzyme tandem repeat drives pentacyclic triterpenoid saponin diversity formation

Fig. 3 Structural diversity and biosynthetic pathways of dragon’s tooth saponin

In summary, this study published a high-quality acacia genome, identified key genes involved in differences in the structure of triterpene saponins, established metabolic pathways for pentacyclic triterpenes saponins, and synthesized a variety of aquedustridine saponins from scratch. The above results reveal the evolutionary mechanism of the structural diversity of triterpenes saponin compounds in the Pentaca family, and also provide important enlightenment for understanding the role of tandem duplicate genes in the evolution of plant secondary metabolism, and also provide high-quality genomic support for the comparative genomics and evolutionary biology of medicinal plants of the Pentaca family (Figure 4). In addition, it also has important guiding significance for the “green bio-manufacturing” of the key medicinal ingredients of the basswood.

Fig. 4 Phylogeny of plants in the family Pentacaidae and the formation mechanism of triterpene saponin compound diversity

Professor Li Yuhua of the College of Life Sciences of Northeast Forestry University, Professor Shen Hailong and Professor Zhang Peng of the College of Forestry, and Professor Liu Zhongjian of Fujian Agriculture and Forestry University are the co-corresponding authors of the paper. Associate Professor Wang Yu and Doctoral Candidate Zhang He of the School of Life Sciences of Northeast Forestry University, North Korean student Hyok Chol Ri (Lee Hyuk-chul), and Doctoral Student An Zeyu are the co-first authors of the paper. Professors Xue Zheyong and Xu Zhichao, School of Life Sciences, Northeast Forestry University, and Professor Tsai Wenjie of National Cheng Kung University in Taiwan participated in the study. This research was funded by the National Natural Science Foundation of China (NSFC) Regional Joint Development Key Fund Project (U21A20243) and the State Key Laboratory of Forest Genetics and Breeding Innovation Project.

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