表观遗传修饰剂线性肟酸对桦褐孔菌三萜合成的影响

Effect of epigenetic modifier SAHA on production of triterpenoids in Inonotus obliquus

目的三萜类化合物是药用真菌桦褐孔菌产生的主要次生代谢产物之一,但其在实验室培养条件下积累量较少。旨在研究组蛋白去乙酰化酶(histone deacetylase,HDAC)活性抑制剂线性肟酸(suberoylanilide hydroxamic acid,SAHA)对桦褐孔菌三萜合成的调控。方法液体摇瓶发酵法培养桦褐孔菌,并在培养液中添加SAHA。采用荧光定量PCR测定三萜合成相关基因转录水平,香草醛-高氯酸法测定细胞内和发酵液中三萜的含量。结果SAHA的添加提高了桦褐孔菌体内编码3-羟基-3-甲基戊二酸单酰辅酶A合成酶、3-羟基-3-甲基戊二单酰辅酶A还原酶、甲羟戊酸激酶、二磷酸酯脱羧酶、角鲨烯合成酶和羊毛甾醇合成酶等基因的表达水平。SAHA显著提高了桦褐孔菌菌丝体内和发酵液中三萜的积累量。SAHA诱导下桦褐孔菌菌丝体内三萜积累量达(66.4±5.24)mg/g,明显高于对照组[(22.7±3.3)mg/g],并且胞外三萜的含量由对照组的(30.5±2.7)mg/L提高至(49.3±3.8)mg/L。此外,经SAHA处理后桦褐孔菌胞内三萜清除自由基的能力显著提高。结论SAHA可以作为调节因子激发桦褐孔菌液体培养条件下三萜类化合物的积累。

Objective Triterpenoids are one of the major classes of bioactive secondary metabolites produced by medicinal basidiomycete Inonotus obliquus.However,the accumulation of triterpenoids is less under lab culture conditions.The purpose of this study was to investigate the regulation of the histone deacetylase(HDAC)inhibitor suberoylanilide hydroxamic acid(SAHA)on the biosynthesis of triterpenoids in I.obliquus.Methods I.obliquus was cultured by liquid shake flask in the presence of SAHA.For determining the effects of SAHA on biosynthesis of triterpenoids by I.obliquus,the m RNA expression level of genes encoding 3-hydroxy-3-methyl glutaryl-CoA synthase(HMGS),3-hydroxy-3-methyl glutary-CoA reductase(HMGR),mevalonate kinase(MK),diphosphomevalonate decarboxylase(DPMD),squalene synthase(SS)and lanosterol synthase(LS)by real-time PCR.For assaying the triterpenoids contents in I.obliquus under submerged culture conditions,Vanillin-Perchloric Acid Method was used to determine the production of total triterpenoids either in mycelia or in the culture broth.Results The results showed that the addition of SAHA enhanced the expression levels of hmgs,hmgr,mk,dpmd,ss and ls in I.obliquus.Consistent with the increase of gene expression,the accumulation of total triterpenoids was increased to(66.4±5.24)mg/g for mycelial triterpenoids and(49.3±3.8)mg/L for extracellular triterpenoids,which was obviously higher than(22.7±3.3)mg/g mycelial triterpenoids and(30.5±2.7)mg/L extracellular triterpenoids found in the control cultures.Furthermore,SAHA treatment enhanced the capacity of mycelial triterpenoids to scavenge free radicals.Conclusion In summary,SAHA can be used as a regulator to stimulate the accumulation of triterpenoids of I.obliquus under submerged culture conditions.