Study On The Effect Of Chemical Intervention Of Gene Expression On Metabolites Of Metarhizium Anisopliae

In recent years,a large number of studies have shown that the chemical intervention of gene expression can possibly release the silenced genes and produce new natural products.Fungi are important sources of lead compounds,among which Metarhizium anisopliae is a broad-spectrum insecticidal fungus.During the long-term coevolution with a variety of insects,there must be a lot of genes in M.anisopliae involved in killing insects being silenced,however,there is no systematic research yet.In this study,two types of chemical inhibitors involving epigenetics,DNA methylase inhibitors and histone deacetylase inhibitors,were used to treat M.anisopliae,and the metabolic variation were studied.HPLC-HRMS analysis revealed that there are a large amount of destruxins in the metabolites of M.anisopliae strain Ma73.Databases and literatures enquiring with molecular formulas and ultraviolet spectra,the main secondary metabolites of Ma73 were identified as destruxin E diol,destruxin B2,destruxin E,destruxin A2,destruxin A3,destruxin B,destruxin B1,subglutinol D,metacridamides A and Fungerin.Among them,the7 destruxins have been reported from M.anisopliae,while subglutinol D,metacridamides A and Fungerin which have antibacterial activities,were first discovered in the metabolites of M.anisopliae.Treatment of M.anisopliae Ma73 strain with 10 and 500μM histone deacetylase inhibitor sodium butyrate or SAHA（Vorinostat）can affect the production of secondary metabolites,and 500μM of sodium butyrate or SAHA can very significantly improve yield of the main secondary metabolites of M.anisopliae.In particular,high concentration SAHA can not only significantly increase the production of main secondary metabolites of M.anisopliae,but also produce new compounds.Using preparative and semi-preparative high performance liquid chromatography methods,two pure compounds were prepared from fermentation broth of 500μM SAHA-treated M.anisopliae Ma73.These two compounds were analyzed by high-resolution mass spectrometry and ultraviolet spectroscopy.Database queries revealed that the to moleculas C₁₀H₉NO₂and C₃₆H₅₀N₁₀O₄are possibly new compounds.The 10 and 500μM histone deacetylase inhibitor benzamide had no significant effect on the metabolism of M.anisopliae Ma73 strain,indicating that the fungus may use benzamide as a nutrient.Treatments with 10μM DNA methylase inhibitor 5-aza-c（5-azacytosine）or 5-aza-dc（5-aza-2’-deoxycytosine）can’t make major secondary metabolites of M.anisopliae changed significantly.But 500μM 5-aza-dc or 5-aza-c can vary the amount and ratio of the main metabolites of M.anisopliae.High concentrations of 5-aza-c can inhibit the production of secondary metabolites such as cytokinin,while 5-aza-dc can increase the production of certain secondary metabolites such as destruxin in M.anisopliae.Despite that 5-aza-c and5-aza-dchave a different mechanism of action they can all release some absolutely silenced genes,and produce new molecule C₁₀H₁₉N₅O.The problem is that the yield is too low.Since sodium butyrate and SAHA can very significantly increase the content of destruxins in metabolites of M.anisopliae,which are important insecticidal active substances with also various other biological activities,sodium butyrate and SAHA can be expected to be used in the production of M.anisopliae insecticides to improve insecticidal efficiency of the fungal pesticide or use the fungal metabolites to prepare natural insecticides.