Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense / 中国实验方剂学杂志

Objective: Ganoderma sinense is one of the most famous medicinal fungi in China. Because it is a model medicinal fungus of basidiomycete,the functional identification of its sesquiterpene synthase is of great significance for the biosynthesis and regulation studies of fungal sesquiterpene. Method: A sesquiterpene synthase gene was discovered by mining the genome data of G. sinense. The sesquiterpene's conservative motifs was analyzed through multiple sequence alignment with two identified sesquiterpene synthases of G. sinense and three terpenoid synthases in the Nr database,which have the highest similarity to it. Subsequently,heterologous expression was observed in Escherichia coli,and protein expression was detected by SDS-PAGE. Volatile compounds were collected by solid phage microextraction (SPME) and detected by GC-MS. Result: The enzyme containing sesquiterpene conserved motifs DDXXDE and NSE/DTE were efficiently expressed in E. coli,and 11 sesquiterpenes were synthesized with endogenous FPP as the substrate. The product α-cadinol at 18.6 min was considered to be the main product,and previous studies showed a significant anticancer activity. According to the comparison with chemical standards,three products were identified as γ-muurlene,α-muurolene and δ-cadinene,respectively. Conclusion: The functional identification of multi-product sesquiterpene synthase from G. sinense can promote the study on the mechanism underlying its product diversity,and lay a foundation for the production of rare or novel sesquiterpenes by improving the catalytic activity of enzymes with enzyme engineering technology.

Antitumor fraction from liquid submerged fermentation of Ganoderma sinense / 中草药

Objective To study the antitumor fraction from liquid submerged fermentation of Ganoderma sinense. Methods The antitumor active fractions were extracted from the hyphostroma which was produced by liquid submerged fermentation and their antitumor activities were observed by mice transplant tumor model. The total polysaccharides were tested by phenol-sulphuric acid method and the contents of reducing sugars were measured by DNS method. The cytotoxic activities were tested by MTT method in vitro. Results The optimum fraction was the intracellular soluble precipitation (sample B) by water extracting with alcohol sedimentation. The contents of total polysaccharide and reducing sugar in the optimum active fraction from liquid submerged fermentation of G. sinense were 88.4% and 3.15%, respectively, but no protein. The inhibitory ratios were 63.94% and 58.32% on the growth of H22 and Lewis tumor transplanted in mice by ig administration in the concentration of 40.5 mg/kg, respectively. The IC50 on four tumor cells of A549, LoVo, CEM, and QGY-7703 were 160, 29.28, 45.06, and 37.38 ?g/mL, respectively, which suggested that the active fraction had weak cytotoxic on LoVo, CEM, and QGY-7703. Conclusion The active fraction from liquid submerged fermentation of G. sinense is polysaccharide which has better inhibitory rate on the growth of H22 and Lewis tumor transplanted in mice but their cytotoxicities are weak.

A new triterpene from fungal fruiting bodies of Ganoderma sinense / 中草药

Objective To study the chemical constituents from the fungal fruiting bodies of Ganoderma sinense.Methods Individual constituents,isolated and repeatedly purified on silica gel and Sephadex LH-20 column chromatography,were identified by physicochemical constants and structurally elucidated by spectral methods.Results Four compounds were isolated and identified as: sinensoic acid(1),cerebroside D(2),poke-weed cerebroside(3),hemisceramide(4).Conclusion Compound 1 is a new compound,others are isolated from the fungus of Ganoderma for the first time.