Regulation Mechanism Of Mitochondrial Glutamic Oxaloacetic Transaminase On Mycelial Growth And Secondary Metabolism Of Ganoderma Lucidum

Ganoderma lucidum is an important edible and medicinal fungus,which can produce a large number of bioactive compounds and has high nutritional and health value and economic development prospects.Among them,G.lucidum triterpenoids and polysaccharides are its important active ingredients,which have anti-inflammatory,anti-tumor,regulating immunity and other biological functions.But at present,the content of natural bioactive molecules is low,how to improve the content of these bioactive molecules is a problem worthy of attention.With the publication of the whole genome sequence of G.lucidum and the improvement of genetic transformation system,it has laid a solid foundation for the in-depth study of secondary metabolism of G.lucidum.Transaminase family,which can synthesize most non essential amino acids in organism.As a key enzyme in the malate aspartate shuttle system(MAS),glutamine oxaloacetate transaminase(GOT)can shuttle the reducing power of NADH from cytoplasm to mitochondria,and maintain the normal operation of mitochondrial function.However,few studies have been done in filamentous fungi,especially in large basidiomycetes.The role of GOT in intracellular nitrogen metabolism,growth and development,and secondary metabolism is still unclear.This study identified two differently located aspartate aminotransferases(GOT1 in mitochondria and GOT2 in cytoplasm)and a homologous gene of alanine aminotransferase GPT in G.lucidum for the first time,and constructed GOT1,GOT2,GPT single silent strains and GOT1/2 double silent strain.It was found that the lack of transaminase can affect the content of intracellular glutamate and glutamine,and the transcription level of nitrogen-related genes.In addition,the silencing of GOT1 and GOT1/2 genes inhibited the growth of hyphae.Compared with the wild-type strains,the growth diameters of the above silent strains decreased by about 42.9%and 49.5%,respectively.The subsequent pharmacological and genetic experiments proved that GOT1 can participate regulate the biosynthesis of ganoderic acid,while GOT2 and GPT have little effect on the synthesis of ganoderic acid:after the addition of aminooxyacetic acid(AOA),the pan-inhibitor of transaminase,the content of ganoderic acids(GAs)increased significantly,about 1.4 times that of normal culture conditions,and then GOT1,GOT,GPT and GOT1/2 were detected respectively after silencing,the content of GAs in the cell was found that only the GOT1single-silencing strain and the GOT1/2 double-silencing strain had a significant increase in the GAs content of G.lucidum,which was increased to 1.45 times and 1.48 times that of the wild strain,respectively.However,the content of GAs in GOT2 and GPT single-silencing strains did not change significantly.These results implied that transaminase can affect the metabolism of nitrogen sources in the cell,thereby affecting the growth of mycelium.Among them,GOT1 located in the mitochondria can significantly affect the biosynthesis of GAs.Next,I initially explored the mechanism by which GOT1 affects the biosynthesis of GAs,and found that the level of reactive oxygen species(ROS)was synchronized with the increase in the content of GAs.The silence of GOT1 increased ROS by approximately 1.7times,and the NAC was externally added,one of the ROS’s scavenger,can restore the content of intracellular ROS and GAs to the wild-type level.These results indicate that ROS signal is involved in the regulation of GAs biosynthesis by GOT1.After further exploring the process of ROS accumulation,it was found that after GOT1 silence,the level of mitochondrial ROS increased by about 1.46 times of the wild type,the oxygen consumption rate(OCR)of mitochondria decreased by 29.3%,and the production of ATP was blocked.It is found that the ratio of NADH/NAD~+in mitochondria is down-regulated by about 26.1%,and the mitochondrial electron transport chain(m ETC)is the main pathway responsible for the production of mitochondrial ROS.These results indicate that the mitochondrial function is disordered,implying that GOT1 silent strains can change the redox homeostasis in the mitochondria affects the normal operation of its function,thereby increasing the level of intracellular ROS to regulate the mechanism of GAs accumulation.In addition,I studied another important G.lucidum secondary metabolite-polysaccharide.After adding AOA,we found the content of intracellular polysaccharides(IPS)and extracellular polysaccharides(EPS)have accumulated 1.54 times and 1.92 times respectively under normal conditions.Further experiments were carried out using the four constructed transformants,and it was found that compared with the wild-type strain,the polysaccharide changes in GOT2i and GPTi strains were not significantly different,while the polysaccharide content in GOT1i and GOT1/2i increased significantly and the key enzyme genes increased to the same degree.This indicates that GOT1 may be the main reason for regulating polysaccharide biosynthesis.Subsequent studies found that GOT1 silencing increased the sensitivity of cell wall,and the relative inhibition rate increased by 21.0%under cell wall stress,while the contents of chitin and glucan,the two main components of cell wall,decreased by 23.5%and 38.1%respectively compared with wild-type strains.At the same time,the expression of the GL18553 gene encoding the mannoprotein in G.lucidum in GOT1i was significantly down-regulated by 41.9%.The above studies show that GOT1 plays an important role in affecting mycelial growth,GAs biosynthesis and G.lucidum polysaccharide accumulation,and lays the foundation for the subsequent in-depth study of the regulation mechanism of nitrogen sources on G.lucidum growth and secondary metabolism.