The Regulation Mechanism Of Glycerophosphatide In Ganoderic Acid Biosynthesis Under Heat Stress In Ganoderma Lucidum

Ganoderma lucidum is widely recognized as a medicinal basidiomycetes because of its secondary metabolites with pharmacological activity.To increase secondary metabolic biosynthesis and understand the regulation mechanism are the urgent issues in the further study.Our previous research have showed that heat stress(HS)could result in a significant increase of secondary metabolites ganoderic acid(GA)biosynthesis.However,how organisms respond to temperature increases and then accumulate GA is still poorly understood.As the primary architecture and first barrier of the cell,the cell membrane plays important roles in sensing environmental change,signal transduction and substance metabolism.Therefore,a potential mechanismit is that cell membrane mediated signal transduction maybe involved in the fungal responding to temperature increases and GA biosynthesis.In the present study,we studied on the regulation mechanism of cell membrane glycerophosphatide in the ganoderic acid biosynthesis under heat stress in G lucidum.The mainly results are as follows:1.We found that HS increased GA biosynthesis by approximately 1.7-fold and also significantly increased cell membrane fluidity.The membrane fluidizer benzyl alcohol(BA)treatment led to significant increases in the GA content compared with no BA treatment.The addition of membrane rigidifier dimethylsulfoxide(DMSO)resulted in significant decreases of the GA content.Furthermore,our results showed that addition of the membrane rigidifier DMSO could revert the increased GA biosynthesis elicited by HS.These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis.Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to wild-type(WT)levels by addition of the membrane fluidizer BA.In contrast,GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO.Furthermore,treatment of D9des-silenced strains with HS led to an increase of approximately 1.3-fold in the GA content compared with the non-HS treatment,and both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains.To the best of our knowledge,this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.2.We performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in G.lucidum.Compared to the G.lucidum WT strain under normal growth conditions,significant changes were observed in the membrane lipid contents of the WT strain after HS treatment for 5,15,30,45 and 60 min.In particular,we observed a significant accumulation of phosphatidic acid(PA)upon HS,PA production 15 min HS-treated samples increased significantly approximately 3.0 fold.In contrast,the total levels of phosphatidylethanolamine(PE)in 30 min HS-treated samples decreased significantly approximately 60%.Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D(PLD)hydrolysing PE.Furthermore,we determined the role of PLD and PA in HS-induced secondary metabolism in G.lucidum.Exogenous 1-butanol,which decreased PLD-mediated formation of PA,reverses the increased GA biosynthesis that was elicited by HS.The pld-silenced strains partly blocked HS-induced GA biosynthesis,and this block can be reversed by adding PA.Also,a 50 ?g/100 mL PA treatment led to significant increases in the GA content(approximately 1.3-fold)compared with samples with no PA treatment.Taken together,our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G.lucidum.Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.3.In previous quantitative analysis of lipids showed that phosphatidylinositol(PI)also accumulates after HS in G.lucidum,which hinted inositol phospholipid signal maybe associated with HS signal transduction.Here,we found that the PI-4-kinases and PI-4-phosphate-5-kinases activities were activated,and their lipid products PI-4-phosphate and PI-4,5-bisphosphate were increased under HS.HS led to a significant increase by approximately 3.7-fold at 20 min and 5.6-fold at 10 min in the PI4K and PIP5K activities,respectively,compared with the non-HS treatment.And HS led to a significant increase by approximately 1.3-fold and 8.6-fold in the PI4P and PI-4,5-bisphosphate contents,respectively,compared with non-HS treatment at 20 min.Further experimental results showded that cytosolic Ca2+and GA content induced by HS run down when cells were pretreated with Li+,an inhibitor of inositol monophosphatases,and this rundown could be rescued by PI and PI-4-phosphate.Furthermore,inhibits PI-4-kinases also resulted in rundown of Ca2+and GA content under HS which could be rescued by PI-4-phosphate,but not PI.However,the rundown of Ca2+and GA content by silencing of PI-4-phosphate-5-kinases could not be rescued by PI-4-phosphate.Take together,our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in cytosolic Ca2+signaling and GA biosynthesis under HS.