The Effects Of Several Common Epigenetic Modifiers On The Growth And Secondary Metabolites Of Monascus Ruber M7

Monascus spp.is one of the important edible microbial resources.Its fermented product of rice,named red fermented rice(RFR),has been widely used in brewing,food coloring and medicines for nearly 2000 years in China and many other Southeast Asian countries.Monascus can produce abundant secondary metabolites,such as Monascus pigments(MPs)and cholesterol lowing agent Monacolin K.However,it can also produce a kind of toxic named citrinin.Analyses of fungal genomes reveal that many gene clusters in fungi that encoding secondary metabolites remain unexpressed under laboratory culture conditions,which are also referred to as silent gene clusters.Studies have shown that some chemical epigenetic modifiers mainly histone deacetylase(HDAC)and DNA methyltransferase(DNMT)inhibitors can alter the epigenetic features of fungal gene clusters,and activate the expression of silent gene clusters,thus induce new fungi metabolites.This study is to focus on the effect of several epigenetic modifiers on development and secondary metabolites of Monascus ruber M7.We have also explored the effect of HDAC inhibitor DHC on the expression levels of different Monascus PKS genes.The main findings are as follows:1.The effects of several epigenetic modifiers on colony and microscopic morphology of M.ruber M7We selected six common epigenetic modifiers(HDAC inhibitor:DHC,SB,SAHA,NT,TSA);DNMT inhibitor:5-AZA).Colony diameters decreased with increasing dose of DHC?SAHA?TSA?5-AZA.Low doses of NT and SB could stimulate colonial growth,whereas higher doses inhibited colonial growth.When the concentration of DHC is above 2mM,the colony is smaller with wrinkle in the center and less aerial hyphae.When the concentration of SAHA is above 200?M,the colony becomes irregular with lighter color.Colonial sizes significantly decreased when the concentration of TSA,5-AZA reach to 2?M.After treated with TSA,the colony folded in the center with less aerial hyphae.After treated with 5-AZA,the colony became less irregular.SB didn't have a significant influence on the development of M7,only on high concentration(>5mM),the colony becomes darker with reduced aerial hyphae.When the concentration of NT is above 10mM,crimson attachments appeared in the colony center.Microscopic morphology results show that M7 grows well and produces conidia and cleistothecia on the plates containing 0.5mM DHC,0.5mM SB,50?M SAHA,0.5mM NT,2?M 5-AZA with no significant change in mycelium morphology.2.The effects of several epigenetic modifiers on biomass,pigments and citrinin production of M.ruber M7 in liquid state fermentation.Within concentrations of SB(5mM),SAHA(200?M),NT(2mM),5-AZA(2?M),the biomass of M7 gradually increased,whereas higher dose inhibited its growth.The biomass of M7 decreased with increasing concentration of DHC,TSA.DHC(2mM),TSA(5?M)significantly influenced the growth of M7.Production of pigments in mycelia gradually increased while in fermentation broth decreased when the concentration of DHC,SAHA is within 5mM,200?M respectively.Production of pigments in mycelia and fermentation broth both increased when the concentration of SB,5-AZA is within 5mM,20mM respectively.Lower dose of NT stimulated red pigments production of M7,while higher dose(8mM)inhibited pigments production.The citrinin production of M7 in fermentation broth decreased with the increasing concentrations of these six epigenetic modifiers,among which DHC,NT,TSA significantly inhibited citrinin production.3.The effects of several epigenetic modifiers on secondary metabolites of M.ruber M7No new secondary metabolite was found after M7 treated with DHC,SB,SAHA,NT,TSA,5-AZA.Production of four kinds of strong blue fluorescent comp1ounds namely BF1-1(m/z=416,C23H28O7),BF1-2(m/z=444,C25H32O7),BF2-1(m/z=374,C21H26O6)and BF2-2(m/z=402,C23H30O6)increased when M7 treated with DHC.BF2-1 and BF2-2 were identified by Balakrishnan(2014)as Monasfluol A and Monasfluol B;BF1-1 was identified by Wu(2013)as Monascusazaphilone C which was separated from M purpureus BCRC 38108.By far,there was no report about BF1-2,so we name it as 12-OAc-monasfluol B.4.The effect of HDAC inhibitor DHC on the expression of different PKS genes of M.ruber M7The effect of DHC on the expression of different PKS genes of M7 was tested by using real-time RT-PCR.The expression level of GME2757 which is highly homologous with citrinin biosynthetic gene in M.purpureus decreased while the expression level of other PKS genes had no significant change.This result coincides with the result that citrinin production decreased after M7 treated with DHC in chapter III.