Lignans are a large group of chemical compounds occurring widely in the plant kingdom. According to the Encyclopedia of Food Sciences and Nutrition, lignans are phenolic dimers with a 2,3-dibenzyl butane structure that mainly occur in the glycosidic form. Some examples of lignans are pinoresinol, podophyllotoxin, and steganacin. As Simpson D. et al. stated, lignans are divided into several categories based on their molecular architecture. Categories of lignans include arylnaphthalene, aryltetralin, dibenzylbutane, dibenzylbutyrolactone, tetrahydrofuran, and furofuran. The interesting structure of lignans gives rise to many bioactive properties that can have high medical values.
In recent years, scientists have become increasingly interested in the health benefits of lignan due to its various bioactive properties. The focus of our research is on the antiviral effects of lignan. In the past, experiments were done on a lignan called NDGA, which proved it could fight the influenza virus by suppressing the replication of IAV and the induction of cytokines, trypsin, and MMP-9. There are many other uses of lignans, including decreasing the risk of cancer and inhibiting inflammation and oxidative injury. What’s more, our research focuses on the antiviral effects of lignan. Zhong Nanshan's group found that lariciresinol-4-O-β-D-glycopyranoside (L4G) could inhibit the inflammatory response induced by the influenza virus, and LDG lariciresinol-4,4'-bis-O-β-D-glycopyranoside had stronger antiviral activity than L4G, which inhibited the release of early viral ribonucleoproteins. Despite its countless health benefits, the use of lignans is strongly limited by the scarce number of grown plants, ineffective treatment methods, and low purity levels of the biosynthesized product.
According to the previous study, lignan biosynthesis is initiated by the formation of pinoresinol, the central precursor of 8–8'-linked lignans, which is then catalyzed by a dirigent protein. After that, the diversity of lignans is increased by glycosylation modifications catalyzed by glycosyltransferases (UGTs). UGTs play key roles in the stability, water solubility, detoxification, and transportation of the plant’s secondary metabolites. However, only a few lignin-related UGTs have been characterized so far, especially the biosynthetic pathway of the antiviral active substance lignan glycosides in I. indigotica has not been fully characterized, which greatly limits the medicinal prospects of lignan glycosides as antiviral active substances. Based on the above research background, this project proposes to discover the UGTs involved in the synthesis of lignan glycosides in I. indigotica by constructing a phylogenetic tree and using synthetic biology to synthesize them heterologously and efficiently, which will be of great significance to improve the production of lignin glycosides.
Figure 1 Work flow of our project
Our group is committed to developing a more efficient and stable method to produce lignan glycosyltransferase, a component involved in the biosynthesis of lignan glycosides, through synthetic biology for sustainable in vitro production. Lignans are a class of naturally occurring active components with antiviral activity and glycosylation modifications are important for the structure and bioactivity of lignans. UDP-dependent glycosyltransferases (UGTs) play a major catalytic role in the glycosylation of woad lignans. Therefore, by means of phylogenetic tree analysis, we found the gene sequence of lignan glycosyltransferase in Isatis indigotica, and UGT72B2 was identified as the lignan UGT in our study.
In the following experimental work, we will use the substrate catalytic specificity of UGT72B2 to identify it after gene cloning. Subsequently, the gene sequence of UGT72B2 is combined with the appropriate vector, and the vector is constructed by Escherichia coli.
The protein expression was induced by IPTG, and the protein expression was detected by SDS-PAGE. At the end of the experiment, we will conduct in vitro enzyme activity experiments by selecting appropriate substrates and conducting product detection.
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