| Ganoderma lucidum,a traditional Chinese medicinal mushroom,has been used for the treatment of various human diseases for several thousand years.G.lucidum produces various natural bioactive compounds,such as ganoderic acids(GAs),polysaccharides and sterols.GAs exhibit various significant pharmacological activities including anti-tumor,anti-metastasis and anti-HIV properties,etc.However,low production of GAs has become a bottleneck for its preclinical researches and commercial application.Therefore,it is necessary to develop an efficient strategy for hyperproduction of GAs in G.lucidum.Biosynthesis of secondary metabolism is usually associated with cell differentiation or development in some fungi.For example,the asexual sporulation affects the biosynthesis of secondary metabolites such as sterigmatocystin,trehalose and aflatoxin in Aspergillus nidulans and A.flavus.In a previous report,G.lucidum differentiates in liquid static culture by forming asexual spores,and this differentiation process is accompanied by higher production of GAs.However,there is no reports on improvement of GA production by regulating asexual sporulation of G.lucdium.In this work,we found that numbers of asexual spores produced by some monokaryons are higher than that of dikaryons in liquid static culture of G.lucidum.Total of 2.49±0.55,2.55±0.21 and 3.14±0.59×10~7asexual spores per cm~2were obtained in the monokaryons G.260125 on day 6,9 and 12 in the liquid static culture condition,which were 1.51,1.51 and 1.34-folds than those in the dikaryon CGMCC5.26,respectively.In addition,the expression level of gene gl25098 in the monokaryons G.260125 were 2.2 and 6.2 times than that in the dikaryon CGMCC5.26on days 6 and 9,respectively.The above results indicate that the asexual sporulation may be promoted in the G.lucidum CGMCC5.26.The cell growth and GA accumulaltion were also investigated in the monokaryons G.260125 and dikaryon CGMCC5.26.Our results showed that the maximum biomass of G.260125 and CGMCC5.26 were 11.07±0.47 and 9.39±0.18 g/L in the liquid static culture condition.The contents of GA-Mk,GA-T and GA-Me and total GAs in the G.260125were up to 78.32±6.93,358.97±33.33,12.75±1.06?g/100 mg dry weight(DW),and 2.44±0.13 mg/100 mg DW,which were 2.9,3.4,1.7,and 1.5 times of those in the CGMCC5.26,respectively.In order to unravel the underlying mechanism of the hyperproduction of GAs,we determined the accumulations of intermediate metabolites(lanosterol and squalene)and the transcription levels of several key genes of GA biosynthetic pathway in the both strains.The maximum accumulated amounts of lanosterol and squalene were 8.49±0.17 and 34.86±6.41?g/100 mg DW in G.260125.These values were 2.4 and 1.8 times higher than those in CGMCC5.26.The transcription levels of 3-hydroxy-3-methylglutaryl-acetyl-Co A(hmgr)and lanosterol synthase gene(ls)in G.260125 were up-regulated by 2.7 and 1.5-folds compared with those in the CGMCC5.26.Our results suggested that the increased of GA contents may be related to the higher accumulation of intermediate metabolites and the upregulated transcription levels of the biosynthetic genes.The developed strategy was also applied in liquid static culture of three Ganoderma strains CGMCC 5.249,5.616 and 5.542.The results revealed that the number of asexual spores in the monokaryons G.5.542-305,G.5.616-1 and G.5.249-500 was 1.54,2 and 1.51 times than that in dikaryon,respectively.The levels of GA-T,GA-S and GA-Me and the content of total GAs in liquid static culture of monokaryon of G.5.249-500 were 9.58,4.47,2.15 and 1.73-folds higher than those in CGMCC5.249,respectively.The contents of GA-T,GA-S,GA-Me and total GAs were also significantly improved in the monokaryon G.5.542-305 and G.5.616-1compared with those in the dikaryon CGMCC 5.542 and CGMCC5.616,respectively.The above results indicate that the developed strategy can be widely used for enhancing GAs production in Ganoderma species.The monokaryons G.260125 and the dikaryon CGMCC5.26 was chosen to study whether the strategy is also effective when the culture system was scaled up from 50 m L to 1L in the liquid static culture conditions.The maximum biomass of G.lucidum monokaryon G.260125 and dikaryon CGMCC 5.26 were 13.46±0.4 and 11.07±0.47 g/L,respectively.The maximum production of GA-Mk,GA-T and GA-Me and total GAs of monokaryon G.260125 were 8.66±0.37,51.72±3.22,1.66±0.08 mg/Land 353.32±18.15 mg/L,which were 2.06,2.18,1.88,and 2.04-folds higher than those in CGMCC5.26,respectively.In this study,we developed a strategy to increase production of GAs by promoting asexual sporulation of Ganoderma species.The high yield of GAs is related to the higher accumulation of squalene and lanosterol and the up-regulation of synthetic genes hmgr and ls.This study provides a new method for the efficient production of GAs and important information for further understanding the relationship between the asexual sporulation and the regulation of GAs biosynthesis in G.lucidum. |
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