Gene Clone And Biochemical Study Of The Key Enzyme For Steroidal Saponin Biosynthesis Of Anemarrhena Asphodeloides Bunge

Objective: The rhizome of Anemarrhena asphodeloides Bunge is a traditional Chinese medicine.It's main pharmacologically active ingredients are timosaponin and its aglycones,and the total account is about 6%.There are many compunds which nearly more than 50 kinds and rich contents.At present,timosaponin BII compounds which is a potential anti AD drug have obvious effects on Alzheimer's disease(AD)and improving learning and memory ability,and has been deeply studied.However,its specific biosynthetic pathways and key enzyme genes are still controversial.The phytosterol saponin relies on phytochemical separation not only is costly,but also subject to natural medicinal resources,and the quality of the extract is difficult to control.Therefore,the purpose of this study was to explore the key enzyme genes in the biosynthesis pathway of timosaponin,and to verify the 2,3-oxidosqualene cyclase(OSC)and sterol side chain methyltransferase 1(SMT1)and Sterol side chain reductase enzyme 2(SSR2)function of Anemarrhena asphodeloides Bunge,It was tested by heterologous expression and biochemical function,which laid a foundation for the study of thesynthesis pathway of timosaponin.Methods:(1)RNA-seq sequencing of the rhizome and aerial parts of Anemarrhena asphodeloides Bunge,collecting all the gene sequences in the steroidal alkaloid synthesis pathway reported in tomato and transcriptome data of other species which mainly contain steroidal saponins.Through Blast to find all the Unigenes associated with the biosynthesis of timosaponin.Then through online tools for gene open reading frame analysis to find full-length and non-full-length sequences.Unigenes related to the synthesis of timosaponin nucleosides were analyzed by qRT-PCR,and the candidate genes of the steroidal saponin synthesis pathway were further predicted by combining the expression characteristics of the candidate genes with the distribution characteristics of the main components of Anemarrhena asphodeloides Bunge.(2)Cloning AaOSC,AaSMT1 and AaSSR2 Candidate Genes from Anemarrhena asphodeloides Bunge Linking AaOSC to yeast expression vectors pESC-URA and pESC-HIS and tobacco expression vector pEAQ-HT-DEST1.AaSMT1 and AaSSR2 were ligated into yeast expression vector pESC-HIS for heterologous expression.The expression products were analyzed by GC/MS.(3)AaOSC and AaSMT1 were compared with homologous genes of other species by MEGA software,The phylogenetic tree of cycloartenol synthase(CAS)and sterol side chain reductase(SMT)was constructed to analyze the evolutionaryrelationship with other species.(4)Using the synthetic biological multi-fragment yeast chromosome homologous recombination technology,the key enzyme gene in the 2,3-oxidized squalene synthesis pathway and AaOSCR12 were integrated into the yeast genome for stable expression.Construction of engineering bacteria producing 2,3-squalene oxide and cycloastrol.Results:(1)A total of 40 Unigenes related to the biosynthesis of saponins from Anemarrhena asphodeloides Bunge were identified by transcriptome sequencing in rhizomes and aerial parts,of which 18 Unigenes were related to the synthesis of saponins.(2)Analysis of the differential expression level of the genes and the metabolism of the timosaponin and sterol,indicating that the timosaponin accumulated in the roots,and the genes related to its synthesis were also specifically expressed in the roots.The content of steroidal saponins in the rhizome was far Above the content of sterols,That means the metabolic flux is very high in the cholesterol-steroidal saponin pathway.(3)The product of AaOSCR12 is cycloartenol,and another product needs further separation and purification to identify the structure,and it is determined that AaOSCR12 is cycloartenol synthase of Anemarrhena asphodeloides Bunge.AaOSCL6 and AaOSCL10 have no catalytic activity.AaSMT1 catalyzes cycloalcohol to 24-methylenecycloartanol with sterol side chain transferase 1 activity.(4)Construction of 2,3-oxidized squaleneengineering strain Y2-SE-10 and cycloartenol-producing engineering bacteria Y3-AaOSCR12.Engineering strain Y2-SE-10 can provide high-content substrates for functional verification of 2,3-oxidosqualene cyclase,and can also be used as the dominant chassis for further gene integration.Engineering strain Y3-AaOSCR12 can increase the production of cycloartenol by 7.4 times,so as to provide substrate support for the function of AaSMT1 and ASSR2 genes.Conclusion: In this thesis,the key enzyme genes on the biosynthesis pathway of timosaponin were discovered,and three AaOSC genes,one AaSMT1 gene and two AaSSR2 genes were cloned and identified.Heterologous expression verified that AaOSCR12 is an cycloartenol synthase.AaSMT1 catalyzes cycloartenol to produce24-methylenecycloartanol with sterol side chain transferase 1 activity.In addition,engineering strains Y2-SE-10 producing 2,3-squalene oxide and Y3-AaOSCR12 producing cycloastrol were constructed,which laid a foundation for the analysis of biosynthesis pathway of saponins from Anemarrhena asphodeloides Bunge and the study of catalytic mechanism of cycloartenol synthase.