Identification And Investigation Of Genes Involved In The Biosynthesis Of Phlorizin And Trilobatin In Lithocarpus Litseifolius(Hance) Chun

Lithocarpus litseifolius（Hance）Chun is an evergreen tree of Fagaceae,which is mainly distributed in the mountains south of the Yangtze River in China,India and Thailand.Tender leaves can be directly decocted into tea soup,long-term use is beneficial to health.The main components of Lithocarpus litseifolius（Hance）Chun are flavonoids and triterpenoids.Phlorizin and trilobatin belong to dihydrochalcone compounds,which are the main active components in Lithocarpus litseifolius（Hance）Chun.They have hypoglycemic,hypolipidemic,anti-inflammatory and anti-tumor biological activities.In addition,Trilobatin is also a healthy natural sweetener,and sweetness is 300 times that of sucrose.However,the natural growth of Lithocarpus litseifolius（Hance）Chun is scattered,and the tree is tall,difficult to pick leaves,time-consuming and laborious,which is not conducive to the extraction of active ingredients.With the development of synthetic biology technology,based on the discovery of efficient key enzyme genes in the biosynthesis pathway of phloridzin and trilobatin,the construction of cell factories to achieve heterologous production of related compounds has become a new sustainable production method.At present,the biosynthesis pathways of phlorizin and trilobatin have been basically elucidated in plant Malus pumila.Several glycosyltransferases have been reported to catalyze the glycosylation of phloretin,but most of them have the problems of low catalytic activity and weak specificity.Therefore,further identification of glycosyltransferases with higher catalytic activity and stronger specificity from plants is an important prerequisite for improving the biosynthesis yield of phlorizin and trilobatin.The contents of phlorizin and trilobatin in Lithocarpus litseifolius（Hance）Chun are abundant.However,there are no reports on the genes involved in the biosynthesis pathway of phlorizin and trilobatin in this plant.Based on this,the high-throughput full-length transcriptome of Lithocarpus litseifolius（Hance）Chun was sequenced in this study.The cloning and functional study of key enzyme genes in the biosynthetic pathway of phlorizin and trilobatin in Lithocarpus litseifolius（Hance）Chun were carried out in order to find the key enzymes that can efficiently catalyze the formation of phlorizin and trilobatin.The main research contents are as follows:1.Determination of phlorizin and trilobatin in different tissues of Lithocarpus litseifolius（Hance）Chun UPLC-Q-TOF-MS was used to quantify the contents of phlorizin and trilobatin in tender leaves,old leaves,tender stems,old stems,roots and rhizome mixtures.The results showed that the contents of phlorizin in different tissues of Lithocarpus litseifolius（Hance）Chun were old leaves,young stems,rhizome mixture,old stems,young leaves,roots from high to low,while trilobatin was only accumulated in leaves,and the accumulation in young leaves was twice as much as that in old leaves.The accumulation of phlorizin and trilobatin in Lithocarpus litseifolius（Hance）Chun provides a stable and reliable experimental material for the screening of phlorizin and trilobatin synthesis pathway genes,and it′s accumulation rule provides a reference for the development of transcriptome sequencing strategy.2.Transcriptome sequencing and gene annotation results of different tissues of Lithocarpus litseifolius（Hance）Chun Results based on compound content detection,the roots,stems,old leaves and young leaves of Lithocarpus litseifolius（Hance）Chun were sequenced by high-throughput sequencing technology.After assembly,splicing and de-redundancy,a total of 34,215 Unigenes were obtained.A total of 33,669 genes were annotated in NR,Swiss Prot,KEGG,KOG,GO,NT,Pfam databases.According to the annotation information,A total of 3 PAL genes,15 4CL genes,35 DBR genes,2 CHS genes and 135 UGT genes involved in the biosynthesis of phlorizin and trilobatin were screened from the transcriptome,which provided a basis for the full-length cloning of subsequent genes.3.Cloning and bioinformatics analysis of double bond reductase Twelve candidate double bond reductase genes were screened from the transcriptome,named Ll DBR1～Ll DBR12.Specific amplification primers were designed and 12 full-length sequences were cloned by PCR.The prokaryotic expression vector p ET28a-HIS-MBP was constructed,and 14 DBR gene sequences were obtained after sequencing analysis.Among them,4 gene sequences were obtained by Ll DBR9 sequencing,and 2 gene sequences were obtained by Ll DBR11 sequencing.Bioinformatics analysis of 14sequences showed that the length of ORF region of Ll DBR1～Ll DBR12 was between1000～1070 bp,and the number of encoded amino acids was between 340-360.Subcellular localization showed that 14 proteins were mainly localized in the cytoplasm.Multiple sequence alignment analysis showed that 14 proteins had conserved GXXS region and glycine-rich region（AXXGXXG or GXXGXXG）.The phylogenetic tree showed that the 14 proteins were clustered with the proteins with reported functions,and the sequence consistency was high.It provides a theoretical basis for the functional identification of later genes.4.Cloning,functional verification and biochemical characterization of glycosyltransferase Twenty-four candidate glycosyltransferase genes were screened from the transcriptome,named Ll UGT1～Ll UGT24.Specific primers were designed and20 full-length sequences were cloned by PCR.The p ET28a-HIS-MBP prokaryotic expression vector was constructed,and 21 candidate UGT genes were finally obtained,of which 3 gene sequences were obtained by Ll UGT9 sequencing.Heterologous expression and in vitro enzymatic reaction showed that Ll UGT4,Ll UGT7,Ll UGT8,Ll UGT9-1,Ll UGT9-2,Ll UGT9-3 and Ll UGT15 could catalyze phloretin to generate glycoside products.The corresponding bioinformatics analysis of these seven proteins was carried out.Ll UGT8 and Ll UGT9-3,which can efficiently catalyze phloretin to phloridzin and trilobatin in vitro,were selected for enzymatic reaction kinetics analysis（the vector was replaced with p ET28a）.The results showed that the optimum reaction time of Ll UGT8 was 8 h,the optimum temperature was 50℃,and the optimum p H value was 8.8 Na₂CO₃-Na HCO₃ buffer system.The optimum reaction time of Ll UGT9-3 was8 h,the optimum temperature was 40℃,and the optimum p H was 8.8 Na₂CO₃-Na HCO₃buffer system.The K_m value of Ll UGT8 was 47.54μM,K_cat was 0.02 s^-1,K_cat·K_m^-1 was365.97 mol^-1·s^-1.the K_m value of Ll UGT9-3 was 79.92μM,K_cat was 0.04 s^-1,K_cat·K_m^-1was 441.06 mol^-1·s^-1.The substrate spectrum screening results showed that Ll UGT8could catalyze the glycosylation reaction of six different structural types of flavonoids with UDP-Glc as the glycosyl donor.Ll UGT9-3 can catalyze the glycosylation of 15different structural types of flavonoids with UDP-Glc as the glycosyl donor.This indicates that Ll UGT9-3 has higher substrate heterogeneities than Ll UGT8.The comprehensive analysis of the biochemical characteristics of glycosyltransferases provides a theoretical basis for the modification of enzymes,and also lays a foundation for the subsequent functional studies in Lithocarpus litseifolius（Hance）Chun.In this study,12 DBR genes and 24 UGT genes involved in the biosynthesis of phlorizin and trilobatin were obtained based on the transcriptome data analysis of Lithocarpus litseifolius（Hance）Chun.Seven glycosyltransferases that can catalyze the glycosylation of phloretin were successfully identified,and two of them can efficiently catalyze phloretin to phlorizin and trilobatin in vitro.Further,the biochemical characteristics of these two glycosyltransferases were studied in details.This study laid a foundation for the production of phlorizin and trilobatin by synthetic biology.