Study On The Molecular Mechanism Of ATP Synthase Mediated Production Of ATP In Triterpene Synthesis Effect In Ganoderma Lucidum

Ganoderma lucidum is a fungus with medicinal value.Triterpenes are one of the main active ingredients in G.lucidum also known as Ganoderic acids(GAs).However,the basic research of G.lucidum is not in-depth at present which limits the development of the G.lucidum fermentation industry.In recent years,the whole genome of G.lucidum has also been reported,allowing Ganoderma lucidum to conduct more detailed molecular mechanism research.Adenosine triphosphate(ATP)as the important "energy currency" in biology is almost involved in all life processes.ATP synthase distributed on the inner mitochondrial membrane of all eukaryotes is the key synthase for eukaryotes to synthesize ATP.ATP plays a very important role in regulating cell metabolism and various other life processes in animal and plant related researchs,but similar studies are rare in fungus and even microorganisms.This research found that Heat stress(HS),Methyl jasmonate(MeJA),Salicylic acid(SA),and Nitrogen starvation treatment could decrease ATP content by 60.0%,74.5%,52.0%and 67.0%respectively in G.lucidum,and increased GAs content by 83.5%,68.0%,31.9%and 35.5%respectively.On this basis,exogenous addition of 2 μM oligomycin(Oli)treatment of WT reduced ATP content by 91.1%and GAs content increased by 66.5%.Exogenous addition of 4 mM adenosine(Ade)treatment of WT increased ATP content by 102.0%,GAs content decreased by 53.5%.Therefore,it is speculated that the change of intracellular ATP will affect the synthesis of GAs in G.lucidum according to the above results.It has practical production significance that GAs content increasing was caused by G.lucidum intracellular ATP content decreasing,we continued to explore its specific molecular mechanism.First,this study identified and cloned the ATP synthase β subunit named it GlATPsyn-β in the G.lucidum’s genomic library.The gene has a total length of 1,626 bp and encodes a 57.96kDa protein with 542 amino acids.Two silent transformants with GlATPsyn-β silencing efficiency of 53.3%and 62.7%named ATPsyn-beta-li and ATPsyn-beta-12i respectively were constructed using RNAi technology in this study.The content of ATP decreased by 69.3%and 75.2%and the content of GAs increased by 158.7%and 142.1%respectively in the two transformants when GlATPsyn-β was silenced.In order to explore the mechanism of GAs content increase caused by the decrease of intracellular ATP content in G.lucidum at the metabolic level,this study examined the content change of acetyl-CoA which was the initial substrate in GAs synthesis.The results showed that the content of acetyl-CoA increased significantly when the content of ATP decreased in G.lucidum.To explore the source of the rise of acetyl-CoA,we examined the changes of genes and enzyme activities both that were the main sources of acetyl-CoA related to glycolytic pyruvate decarboxylation and fatty acid β oxidation,and finally found that the expression of genes and enzyme activities related to fatty acid β oxidation were significantly increased.In order to prove that fatty acid β oxidation was involved in the increase of GAs content caused by the decrease of intracellular ATP content in G.lucidum.This study constructed double-silent strains named ATPsyn-ACD and ATPsyn-HCD of ATP synthase β subunit and fatty acid β oxidation key enzymes,and used 10 μM fatty acid βoxidation inhibitor etomoxir(eto.)to treate GlATPsyn-β silenced strain for pharmacological experiment comparison.The results indicated that the GAs content of ATPsyn-ACD,ATPsyn-HCD double-silenced strains and use 10 μM eto.to treate GlATPsyn-β silenced strains decreased at least 35.9%compared with GlATPsyn-β silenced strains,and there was no significant difference compared with WT strain.At this time,the content of acetyls-CoA in ATPsyn-ACD and ATPsyn-HCD double-silent strains was also decreased at least 51.5%compared with GlATPsyn-β silenced strains,and there was no significant difference compared with the WT strain.Therefore,this study suggested that at the metabolic level,fatty acid β oxidation participates in the process of GAs content increase caused by the decrease of intracellular ATP content.In eukaryotes,changes in ATP levels often affect the content of intracellular second messenger cAMP.In order to explore the mechanism of GAs content increase caused by the decrease of intracellular ATP content in G.lucidum,this study examined the changes of substrates and enzyme activities in cAMP-PKA pathway when ATP content decreased in G.lucidum.The results implied that the intracellular cAMP content was increased by 316.2%and 296.1%respectively,and the enzyme activity of PKA was increased by 53.2%and 50.9%respectively of two GlATPsyn-β silent strains.In order to prove that the cAMP-PKA pathway was involved in the decrease of intracellular ATP content resulted in the increase of GAs content in G.lucidum.A double-silent strain of ATP synthase β subunit and PKA catalytic subunit named ATPsyn-PKAc was constructed,GlATPsyn-β silenced strains treated with 20 μM PKA inhibitor H89 used to compare pharmacological experiments.The experimental results showed that the GAs content of the ATPsyn-PKAc double-silent strain and used 20 μM H89 to treat the GlATPsyn-β silencing strain decreased at least 39.3%compared with the GlATPsyn-β silencing strain,and the GAs content level have no significant difference compared with the WT strain.Therefore,the cAMP-PKA pathway was involved in the increase of GAs content caused by the decrease of intracellular ATP content.This study preliminarily proved that the content of ATP would affect the GAs content.Moreover,genetic approachs that silence the gene expression of ATP synthase β subunit to reduce the ATP were used to preliminarily prove that the decrease of the content of ATP would induce the increase of GAs content from the perspective of metabolism and signal,which provided a theoretical basis for the study on the influence of ATP on secondary metabolism in fungi in the future.