Cloning And Function Of CYP450 Gene Involved In The Biosynthetic Pathway Of Pachymic Acid In Poria Cocos

Poria cocos(Schw.)Wolf belongs to Polyporaceae family,its sclerotium is a kind of traditional Chinese medicine in China.The terpenoid pachymic acid with several pharmacological activities is the main medicinal ingredient and herbal quality index component of P.cocos.In production,pinewood is usually used to cultivate P.cocos,therefore the cultivation of P.cocos contradicts the conservation of forest resources.To reveal the synthetic pathway of pachymic acid can help to produce pachymic acid using heterologous synthesis.It will reduce the consumption of pine wood resources.Based on the understanding of the structural characteristics of the triterpenoids identified from P.cocos,a downstream biosynthetic pathway of pachymic acid with lanosterol as precursors was proposed,in which CYP450 enzymes catalyzed multiple sites of hydroxylation or carboxylation reactions.In this study,we combined the analysis of the genomic and transcriptomic sequencing results of P.cocos to mine the key CYP450 enzyme genes involved in the biosynthetic pathway,and qualitatively quantified the related metabolites contents changes in the metabolic pathway after overexpression and silencing of these candidate CYP450 genes in P.cocos,so as to deduce candidate CYP450 gene functions and further deepen the analysis of pachymic acid synthesis pathway.The main results are as follows:1.In this study,the genome of P.cocos was sequenced using the Pac Bio Sequel II sequencing platform.The size of the assembly genome was 58.1 Mb,and 185 contigs were assembled with a contig N50 length of 1419924 bp and a GC content of 51%.There were93% genes(16015 genes)was annotated.2.A total of 298 CYP450 genes were annotated,and all these genes were used to perform gene cluster analysis and gene expression profiling with four genes(LSS,SMT1,SOAT and Pc CYP3)which were revealed involved in the biosynthetic pathway of pachymic acid in P.cocos were taken as reference genes.Fourteen candidate CYP450 genes were screened,and four CYP450 genes,Wc CYP118,Wc CYP158,Wc CYP220,Wc CYP153,with high correlation of gene expression patterns,were selected for the further research.3.Wc CYP118,Wc CYP158,Wc CYP220 and Wc CYP153 were successfully cloned from P.cocos,and sequence analysis showed that Wc CYP158,Wc CYP118 and Wc CYP153 were unstable hydrophilic proteins,and Wc CYP220 was a stable hydrophilic protein.Based on the sequenve alignment,it showed that Wc CYP158 and Wc CYP118 belonged to fungal CYP64-like,Wc CYP220 belonged to CYP450 52 family,and Wc CYP153 probably belonged to CYP FUM15-like.Wc CYP118 had a signal peptide with transmembrane region structure,Wc CYP153 and Wc CYP118 had a signal peptide and transmembrane structure,Wc CYP153 and Wc CYP220 had no signal peptide but had transmembrane structure,and Wc CYP158 had no signal peptide and transmembrane structure.4.The overexpression vectors and silent expression vectors of the four candidate genes were successfully constructed and the corresponding transformants were obtained.The q PCR results revealed that compared with the wild-type strain,the gene expression of the overexpression strains of the four genes were significantly up-regulated and the gene expression of the silent expression strains were down-regulated.Profiling of metabolites in transformants revealed that the contents of pachymic acid,16α-hydroxytrametenolic acid,eburicoic acid,tumulosic acid were increased in the strains with Wc CYP118,Wc CYP153 and Wc CYP220 up expression but in the strains with Wc CYP118,Wc CYP153 and Wc CYP220 were silenced,the contens of pachymic acid,16α-hydroxytrametenolic acid,eburicoic acid,tumulosic acid were decreased.It is obviously that Wc CYP118,Wc CYP153 and Wc CYP220 positively regulated the synthesis of pachymic acid,16α-hydroxytrametenolic acid,eburicoic acid,tumulosic acid,and it is assumed that the above three genes are involved in the biosynthetic pathway of pachymic acid.In summary,this study deduced the pathway of pachymic acid biosynthesis and screened four CYP450 enzyme genes that may be involved in the pathway of pachymic acid biosynthesis based on genomic and transcriptomic data analysis.Through functional validation analysis,Wc CYP118,Wc CYP153 and Wc CYP220 were found to be involved in the pathway of pachymic acid biosynthesis,and the result provided a foundation for further complete elucidation of pachymic acid biosynthesis pathway.