| Ganoderma lucidum is a kind of Chinese traditional herbal medicine for the treatment of diseases including hepatopathy,nephritis,neurasthenia and asthma.Modern research shows that polysaccharide and triterpenes are the main bioactive substances in G.lucidum,which could modulate immuno-ability and inhibit tumor cell growth.At present researchers primarily focus on its medicinal functional and mechanism.However,substrate degradation and accumulation of bioactive substances during growth of G.lucidum are less concerned.Therefore,the G.lucidum G0119,which possesses high spore production and has been commercially cultivated on a large scale in China,was investigated in this study.Based on the changes of lignocellulose,carbohydrates and triterpenes during growth of G.lucidum,using sequencing of genome,transcriptome and secreteome,the correlation between lignocellulose degradation,carbohydrate metabolism and accumulation of bioactive compounds were investigated,aiming to provide new data for the guide of harvest and production of G.lucidum under scientific control.The main conclusions were listed below:(1)Mechanism between lignocellulose degradation and carbohydrate metabolism during growthComparing with carbohydrate active enzymes of other Ganoderma strains or model basidiomycetes strains,strain G0119 possesses more genes of cellulose and hemicellulose degradation.Furthermore,transcriptomic and proteomic analysis by different growth phases indicated that lignocellulose degradation in substrate was found correlated with carbohydrate metabolism during growth of G.lucidum G0119,and the contents also changed significantly.The expression level of ligninase and hemicellulase genes were higher from mycelium growth phase to primordium phase,and the expression level of cellulase genes started increasing.Accordingly,the contents of lignin in substrate decreased from 22.54 % to 19.08 %,cellulose decreased from 21.24 % to 16.71 %,and hemicellulose decreased from 13.86 % to 11.49 %.As the degradation products,Carbohydrate catabolism including EMP,HMP and TCA,also started.Glucose in substrate increased from 0.41 % to 1.43 % at mycelium growth phase,and started decreasing since the primordium formed.These results indicated that after lignin was broken through,hemicellulose and cellulose were degraded.And the products entered into cells to supply energy,reducing power and carbohydrates for development of fruiting bodies.From primordium phase to young fruiting body phase,the expression of cellulase and hemicellulase genes kept increasing.Contents of cellulose and hemicellulose decreased to 16.71 % and 11.49 %.The carbohydrate catabolism was more incline to EMP and TCA to HMP pathway.Increasing of carbohydrate catabolism resulted in decreasing of glucose to 0.52 %.Enzyme expression involving in carbohydrates synthesis such as glycogen,trehalose and polyol,were also increasing in this phase.These results indicated that the degraded carbohydrates not only participated in central metabolism but also synthesis of other carbohydrates after entering into cells,resulting in increasing of trehalose,polyol and polysaccharides to 1.71 %,0.32 % and 1.81 %,which not only possessed physiological function such as stress resistance or stabilizing osmotic pressure,but were also carbon storage for metabolism afterward.At the last phase when the fruiting bodies matured and spores were massively ejected,expression of most cellulase genes were upregulated and the cellulose content decreased to 12.67 %.Increasing of carbohydrates metabolism in mycelium resulted in decreasing of glucose in substrate to 0.24 %.The expression level of TCA was stable in high level.Enzymes involving in synthesis of trehalose and glycogen were downregulated,resulting in decreasing of glycogen accumulated in earlier time.Contents of mannitol and trehalose also decreased to 0.04 % and 0.03 %.These facts indicated carbohydrates metabolism were more inclined to carbohydrate catabolism in this phase.The expression of chitin synthase kept increasing.These results suggested there was a great increasing for the requirement of energy supply and cellular carbon skeleton synthesis from substrate and carbon storage in mycelium for maturation of fruiting bodies and spore production.(2)Correlation between triterpene and carbohydrate metabolism based on lignocellulose degradationTriterpene is synthesized based on acetyl CoA from central carbohydrate catabolism.From mycelium growth phase to primordium phase,enzymes of triterpene upstream synthesis,from acetyl CoA to precursor mevalonate,including ACAT,HMGS and HMGR were expressed in high level.And low-polarity triterpenes were also accumulating.Their contents were correlated with gene expression of cellulase and central carbohydrate catabolism enzymes.From primordium phase to young fruiting body phase,the increasing of cellulase and hemicellulase expression were correlated with expression of carbohydrate catabolism enzymes.Metabolism of EMP and HMP,together with synthesis of high-polarity triterpenes also increased.The increasing of EMP metabolism could provide more acetyl CoA for triterpene precursor synthesis,and NADPH produced from HMP metabolism could increase the enzyme activity involving in triterpene metabolism.Therefore,the increasing of central metabolism enhanced synthesis of triterpenes precursors.Degradation of cellulose and hemicellulose supplied precursors for the upstream synthesis of triterpene from carbohydrate metabolism products.Expression of 4 CYP genes were found correlated with expression of precursor lanosterol synthase.Expression of 1 CYP gene was correlated with high-polarity triterpenes and this gene was highly expressed in this phase.During this phase the content of high-polarity triterpenes maintained a high level,among which ganoderic acid B increased to 1.98 ‰.Low-polarity triterpenes also increased.Meanwhile,the expression of synthases of trehalose,arabinitol,glycogen and contents of them all increased.Contents of trahalose and arabinitol in fruiting bodies increased to 1.71 % and 1.42 %.From mature fruiting body phase to spore fully ejecting phase,contents of high-polarity triterpenes decreased significantly,among which ganoderic acid B decreased to 0.33 ‰.Decreasing of high-polarity triterpenes were correlated with downregulation of MPK,which was also triterpene precursor synthase.Meanwhile,contents of high-polarity triterpenes were correlated with gene expression of cellulase,glycogen catabolism enzyme,suggesting the synthesis of high-polarity triterpenes was decreasing in this phase.Besides of that,there were 3 CYP genes found correlated with changes of low-polarity triterpenes and they were expressed at high level in this phase.Low-polarity triterpenes increased significantly.Content of ganoderic acid T increased to 1.26 %,which indicate that downstream metabolism of triterpene transferred from synthesis of high-polarity triterpenes to synthesis of low-polarity triterpenes.Contents of low-polarity triterpenes were correlated with expression of cellulase,and polysaccharide synthase.At this phase,level of glycogen dropped significantly.Contents of cellulose and trehalose decreased to 12.67 % and 0.25 %.These facts suggested that the increasing of cellulose degradation and catabolism of carbon storage not only supplied carbohydrates and energy for fruiting body maturation and spore production,but also enhanced synthesis of low-polarity triterpenes.(3)The changes of bioactivity compounds in different regions of G.lucidum G0119 during fruitingThe composition of triterpenes and contents were changing during fruiting of G.lucidum G0119.High-polarity triterpenes was decreasing and low-polarity triterpenes was increasing during growth,suggesting the main triterpene synthesis flux downstream was changing from synthesis of high-polarity triterpenes to synthesis of low-polarity triterpenes.The high-polarity triterpenes were accumulated in the stipe the young fruiting body and the base region of the fruiting body mainly produced low-polarity triterpenes.Results indicate that there may be more downstream pathway for high-polarity triterpenes in fruiting bodies than in mycelium of base region during growth of G.lucidum.In summary,correlation between lignocellulose degradation,carbohydrate,upstream metabolism of triterpene precursor and switching of triterpene downstream pathway was analyzed in this study.Based on the results,an improved harvest method of G.lucidum was proposed.The best G.lucidum material for the highest amount of bioactive substances was the young fruiting body.It contained the highest content of β-glucan and high-polarity triterpenes.And the highest content of low-polarity triterpenes was also contained in the base region after harvesting multiple flush of young fruiting bodies.By this harvest method,the total production of young fruiting bodies was even higher than harvesting the mature fruiting bodies using the traditional method.This finding provided scientific data for obtaining higher bioactive substances from Ganoderma materials.And this harvest method will show a wide application prospect in the future. |
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