Heterologous expression and product identification of diterpene synthase involved in the biosynthesis of brasilicardin A
Brasilicardin A,a diterpene glycoside isolated from pathogenic actinomycete Nocardia brasiliensis IFM 0406,has become a novel immunosuppressant candidate due to its significant immunosuppressive activity,low toxicity and unique mechanism of action.However,brasilicardin A and its analogues have become a research hotspot to the development of this promising immunosuppressant because of the low-yield production in the natural pathogenic producer and the synthetically challenging skeleton.According to the reported biosynthetic pathway of brasilicardin A,the function of involved diterpene synthase was analyzed by bioinformatics.Then the genes bra1-5 that synthesize the brasilicardin A skeleton were directionally amplified from the pathogenic strain N.brasiliensis IFM 0406,and heterologous expression was achieved successfully in Streptomyces albus R1.The compounds were isolated and purified by using various column chromatographies including silica gel column chromatography and semi-preparative HPLC.Six new brasilicardins were established and named brasilicardin H-M.The activity of brasilicardins was screened using lipopolysaccharide(LPS)-activated mouse primary macrophage inflammation model.Brasilicardin H-M exhibited good inhibitory activity on nitric oxide(NO)release with IC50 values of 28.24±3.70,37.44±2.00,39.85±4.02,26.77±4.40,65.25±1.48 and 15.24±2.72 μmol·L-1,respectively(indomethacin as the positive control with IC50 value of 34.28±4.10 μmol·L-1).The results indicated that six compounds had potential anti-inflammatory activity.This study laid a foundation for the elucidation of the brasilicardin A biosynthetic pathway and evaluation of the structure-activity relationship as well as new drug developments.
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