Metabolic Distribution And Preliminary Biosynthetic Research Of Kinsenoside From Anoectochilus Roxburghii

Anoectochilus roxburghii（Wall.）Lindl is a perennial herb belonging to the Orchidaceae family and the whole plant is used as a traditional Chinese folk medicine.Kinsenoside which is the major active constituent in A.roxburghii has been well documented to possess significant pharmacological activities such as anti-hypoglycemic,anti-hypolipisis and hepatoprotective.With the widely and constantly exploration of the medical value of kinsenoside,the development of kinsenoside-associated drugs becomes to be a general trend.Till now,the only accessible resource of kinsenoside substance is extraction from A.roxburghii.However,the expensive plant materials,uncertainty of origin and difficult to separate isomer-product,drive up the cost of the product.To solve this problem,we turn to use biosynthetic approach to produce kinsenoside in a heterologous host.Natural product biosynthesis has for long been recognized for its high efficiency and stereoselection,however,the initial step is to illuminated the biosynthetic pathway and the involved genes in the origin host.Firstly,an accurate and sensitive method to determination the concentration of kinsenoside in A.roxburghii by liquid chromatography-triple quadrupole mass spectrometry（LC-QQQ-MS）was established,and then,the metabolic distribution of kinsenoside in different A.roxburghii varieties,different growth periods and different plant tissues were analyzed.The temporal and spatial distribution profiles of kinsenoside in A.roxburghii provided basis to the screen of samples for transcriptome sequencing.Based on these cues,full-length transcriptome sequencing and high-through gene expression level sequencing on the root,stem,and leaf of three months old A.roxburghii were carried out.As a result,12 glycosyltransferase genes were screened out as candidates by a combined analysis of the gene expression level and metabolic profiles of kinsenoside in different tissues.To narrow the screening range of candidate genes for the biosynthesis of the kinsenoside aglycone（R）-3-hydroxy-γ-butyrolactone[（R）-3HBL],¹³C stable isotope tracing approach was applied to investigate the biosynthetic pathway of（R）-3HBL.After feeding with different ¹³C labeled glucose and sodium acetate in the culture media,the product were detected and analysis by LC-q TOF-MS and NMR.Besides,with the purpose of creating a tool for the function identification of candidate genes and constructing a kinsenoside producing strain.A[（R）-3HBL]producing E.coli strain named EMG3-C was reengineered by following the procedures presented in previous researches.In summary,the implementation of the above work will lay the foundation for the illumination of the molecular mechanism of the biosynthesis of kinsenoside in A.roxburghii and provide the basis for the engineering of kinsenoside biosynthetic pathway in heterologous cells.