| Poria cocos is a fungus widely used as a Chinese traditional medicine for centuries.The main medicinal effects described to date are the water permeating dampness,strengthening the spleen and strengthening the heart.P.cocos is also used in preparation of different food items(e.g.tuckahoe cake and Tuckahoe wine).The compound responsible for its unique pharmacological properties is believed to be Pachymic acid.Previous researchers have summarized the results of the medicinal activities of Pachymic acid which include anti-tumor,anti-inflammatory,anti-oxidation,hypoglycemic,sedative and hypnotic properties.At present,pachymic acid is mainly extracted from P.cocos.Cultivation conditions largely influence yield of Pachymic acid from P.cocos biomass.Keeping in mind the importance of Pachymic acid,there is a need to developed bio-engineered strains of P.cocos ensuring increased production of Pachymic acid.At present,bio-synthetic pathway of pachymic acid production is unknown.In this study,we speculated the biosynthetic pathway of pachymic acid in Poria cocos for the first time along with identification of key enzymes involved in biosynthetic pathway.In this regard,C-24 methyltransferase was mainly focused and cloned for further studies.Some of the findings of current study are summarized in below:1 In first phase of study,the biosynthetic pathway of pachymic acid was speculated as shown in Figure 1-2.This pathway involves four key enzymes namely;cytochrome P450 s,sterol C-24 methyltransferase(SMT1),sterol acyltransferase(SOAT)and sterol ester hydrolase(TGL4).Lanosterol is a precursor molecule undergoing series of catalytic reactions to be converted into pachymic acid.2 The SMT1 gene was cloned,and two homologues sequences were obtained namely,SMT1-1 and SMT1-2.The open reading frame of SMT1-1 consists on 1038 bp.It contains2 introns,3 exons,and encodes 345 amino acids.Protein encoded by this gene is hydrophilic in nature.The open reading frame of SMT1-2 gene is of 1050 bps.It contains4 introns,5 exons,encoding 349 amino acids.Here also protein is of hydrophilic nature.Protein 3D structure simulation showed that the structure of protein SMT1-1 was highlysimilar to that of SMT1-2.Phylogenetic analysis showed SMT1 homology with Xanthophyllomyces dendrorhous.3 The prokaryotic expression vector pCold-SMT1 was constructed and transformed into E.coli expression strain BL21(Pg-KJE8).The prokaryotic expression condition was standardized,and a large amount of SMT1 protein was expressed.Furthermore,SMT1 protein was used to verify the chemical reaction of trametenolic acid responsible for generation of eburicoic acid.The presumed reaction was verified to be correct.4 Fluorescence quantitative PCR was performed to quantify the expression of SMT1 gene in Poria cocos.The mycelial biomass of Poria cocos was produced by culturing on PDA medium at 26℃.Expression of SMT1 gene was observed in a time course manner(6,9,12,15,and 18 days after cultivation)by performing quantitative PCR.Here different expression levels of SMT1-1 and SMT1-2 gene were seen.The SMT1-1 overall expression is significantly higher than that of SMT1-2.These results suggest that SMT1-1 play a main role in pachymic acid biosynthesis. |
