| Ganoderma lucidum has become increasingly popular as a medicinal macrofungi with varieties of pharmacologically active compounds.So,the artificial cultivation of G.lucidum has also received attention from researchers.During the cultivation of G.lucidum,it is subjected to a variety of stresses such as nutrient stress(inadequate utilisation of carbon sources),heat stress and so on.The mitochondrial pyruvate carrier(MPC)is located in the inner mitochondrial membrane and is an essential carrier for pyruvate to enter the mitochondria.Some researches have indicated that MPC could regulate a wide range of intracellular metabolisms such as glycolysis,the TCA cycle,fatty acid metabolism and amino acid metabolism.The shift in carbon source can regulate the activity of MPC and thus change the metabolism of cells.However,little is known about the metabolic regulatory role of MPC in macrofungi.In this paper,we focused on the metabolic regulatory role of MPC in response to carbon sources and to ganoderic triterpenes synthesis under heat stress,the mainly results are as follows:1.To investigate the relationship between MPC and lignocellulose decomposition in G.lucidum,this research first identified the mitochondrial pyruvate carrier in G.lucidum by homology comparison and found that MPC in G.lucidum is mainly encoded by two genes(GlMPC1 and GlMPC2),which form a dimeric complex for the mitochondrial transport of pyruvate.And,two transformants with transcript levels between 20% and 40% of the wild type were screened by real-time fluorescence quantitative PCR respectively,for subsequent experiments.Then,we constructed GlMPC1 and GlMPC2 knockdown transformants by RNA interference technology.We found that the knockdown of MPC increased the growth rate of G.lucidum about 30% in a medium with wood chips as a carbon source.Then,cellulase and laccase activities were tested and approximately 50% of endoglucanase enzyme activity as well as 35% of laccase activity were increased in MPC knockdown mutants compared to WT.Finally,the expression levels of gene related to glycolysis(HK glucokinase;PFK phosphofructokinase;PK pyruvate kinase)were assayed,and the transcription levels of these enzymes were found to be increased by approximately 250%compared to the WT strain.The above results indicated that knockdown of GlMPC promoted the decomposition rate of lignocellulose by G.lucidum and increased endoglucanase activity and laccase activity,which provided a new idea to improve the decomposition and utilization of non-dominant carbon sources in G.lucidum.2.To investigate the relationship between GlMPC and the metabolic regulation of triterpene synthesis in G.lucidum under heat stress,this research first assayed the transcript levels of GlMPC under heat stress and found that the expression levels of GlMPC1 and GlMPC2 were significantly reduced at 1.5 h of heat treatment,and their expression levels were only 50% and 60% of those of the control group(no heat treatment).Then we examined the ratio of mitochondrial pyruvate content to intracellular pyruvate content in G.lucidum under heat stress and found that at 1.5 h of heat treatment,the ratio was reduced by approximately 25% under GlMPC1 or GlMPC2 knockdown conditions compared to the control.This research assayed the effect of GlMPC on the ganoderic triterpenes content and found that the triterpenes content of the GlMPC1 and GlMPC2 knockdown transformants was increased by approximately 50% compared to WT.In GlMPC1 and GlMPC2 overexpression transformants,the ganoderic triterpenes content decreased by approximately11% compared to the WT.To further investigate the mechanism of GlMPC regulation of triterpenes synthesis in Ganoderma lucidum,we investigated the downstream metabolic regulation of GlMPC.By assaying the expression levels of three key enzymes of the TCA cycle(citrate synthase CS,isocitrate dehydrogenase ICDHm and α-ketoglutarate dehydrogenase α-KGDH)in GlMPC knockdown transformants,the expression levels of CS and ICDHm were found to be increased by approximately 100% under GlMPC silencing compared to WT,while the expression level of α-KGDH did not change significantly.Then,this research examined the enzymatic activity of ICDHm and α-KGDH and found the enzymatic activity of ICDHm was increased by approximately 44% but no significant change in the enzymatic activity of α-KGDH in the GlMPC knockdown transformants.This means that silencing of GlMPC did not significantly down-regulate the TCA cycle.The expression levels of key enzymes of the TCA cycle under heat stress and the enzymatic activities of ICDHm and α-KGDH were assayed in concordance with the results.The substrates of the TCA cycle can be derived from acetyl Co A from mitochondrial pyruvate supply,in addition to acetyl Co A from fatty acid β-oxidative metabolism andα-ketoglutarate from Gln back-completion.Therefore,through the results of the enzymatic activity assays of ICDHm and α-KGDH,we hypothesized that the activity of GlMPC decreased under heat stress,and G.lucidum up-regulated fatty acid metabolism in order to maintain the TCA cycle.This was later verified by the results of the transcript level assay of genes related to fatty acid β-oxidation under GlMPC knockdown transformants and heat stress treatment.Finally,we constructed co-silencing transformants of GlMPC and fatty acid β oxidation and a reduction of approximately 40% in the ganoderic triterpenes content was found in the co-silenced transformants compared to WT.The above results suggest that GlMPC responds to heat stress and enhances ganoderic triterpenes synthesis through up-regulation of fatty acid β-oxidation.In summary,GlMPC not only responds to G.lucidum carbon source utilization and negatively regulates the decomposition rate of lignocellulose,but is also associated with altered metabolism under heat stress and promotes ganoderic triterpenes synthesis by negatively regulating fatty acid β-oxidation.The present study provides new insights into the adaptation of MPC to stress through metabolic regulation of cells in fungi,especially in Basidiomycetes. |
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