Cloning And Functional Characterization Of Glycosyltransferases From Andrographis Paniculata

Andrographis paniculata is an important traditional medicinal plant in South and Southeast Asia.It has the functions of clearing away heat and toxins,cooling blood and detumescence.It can be used for exogenous toxins,fever,sore throat,sore mouth and tongue,and cough.At present,Andrographis paniculata is not only a traditional Chinese medicine,but is also made into a variety of modern dosages for clinical use.Diterpenoid glycosides and flavonoid glycosides are one of the main effective components in A.paniculata.They are usually derived from chemical synthesis or extraction and separation from the original plant,which is difficult to meet the needs of commercialization.Compared with chemical methods,enzymatic methods avoid environmental pollution caused by the use of a large number of organic solvents,and reduce the formation of by-products,which make up for the difficulties to establish chemical synthesis pathways in the synthesis of compounds with complex structures.Glycosylation is an important post-modification and transformation reaction,which widely exists in plants.Glycosylation can transform aglycones into more stable,structurally diverse and biologically active glycosides.Glycosyltransferases are key enzymes involved in the biosynthesis of many active glycosides.There are many glycosyltransferases in plants that perform different glycosylation functions.Cloning and functional characterization of this multi-gene family will be of great significance.It will provide new bases for the enzymatic synthesis of glycosides from natural small molecules,for the exploitation of glycosylation enzymes and the development of new drugs.However,the glycosyltransferases from Andrographis paniculata remain unknown.In this study,a novel diterpenoid glycosyltransferase and three glycosyltransferases with broad substrate spectrums and can be used for multi-site glycosidation of substrates were obtained from Andrographis paniculata.1.Excavation of Glycosyltransferases from Andrographis paniculataTaking neoandrographolide as an index component,the gene expression in the leaves of Andrographis paniculata were analyzed to determine the candidate glycosyltransferases.A total of 9 glycosyltransferases were screened,whose accumulation patterns of neoandrographolide during methyl jasmonate induction were consistent with the expression patterns in transcriptome.Bioinformatics studies of these 9 genes were also carried out.These candidate glycosyltransferase genes were subsequently expressed in prokaryotic expression system.2.Functional characterization of a diterpenoid glycosyltransferase from Andrographis paniculataBy screening the catalytic functions of the nine glycosyltransferases from Andrographis paniculata,we successfully identified a glycosyltransferase(Ap UGT5)that showed catalytic ability of glycosylating andrograpanin.The structure of the glycosylated product was identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,1H NMR and 13 C NMR spectroscopy.Ap UGT5 is a novel diterpenoid glycosyltransferase with broad substrate spectrum and high catalytic efficiency.It can catalyze the C-19 hydroxyl group of andrograpanin to form neoandrographolide,and can catalyze 26 different types of substrates,including diterpenoids and their derivatives,triterpenes,flavonoids,alkaloids,phenylpropanoids,coumarins and simple aromatics with various nucleophilic groups(-OH,-SH,and-NH2).3.Functional characterization of Flavonoid Glycosyltransferases from Andrographis paniculataThree glycosyltransferases with catalytic activity for flavonoids were also identified.The structure of glycosylated products were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.The enzymatic reactions were carried out with flavonoids,flavonols,flavanone,isoflavones,dihydrochalcone,coumarin and other small molecular aromatic compounds with-OH and-NH2 groups as substrates.For the same substrate,the types and conversion rates of glycosylated products catalyzed by the three glycosyltransferases are different.For some flavonoids,these three glycosyltransferases all exhibit strong position selectivity,producing only one glycosylation product,while for other flavonoids,they can be converted into different multi-site glycosylation products.In conclusion,the glycosyltransferase homologous genes were screened based on the transcriptome data of A.paniculata leaves.A diterpenoid glycosyltransferase and three flavonoid glycosyltransferases with broad substrate spectrums and high catalytic efficiencies have been successfully identified.They can be used as good tools for the enzymatic synthesis of bioactive glycosides with different structures,providing molecular basis for the glycosylation mechanism of diterpenoids and flavonoids,promoting the studies on structural diversity of drug-leading compounds,and providing evidences for further application of enzymatic engineering and modification of compounds by enzymatic or synthetic biology.