Effects And Molecular Mechanism Of Reactive Oxygen Species On The Growth And Lipid Production Of Chlorella Pyrenoidosa

Human society are facing two major problems that are energy crisis and greenhouse effect.Microalgal biodiesel achieve energy output and carbon emission reduction simultaneously,which is a hot research topic in recent years.Under suitable conditions,microalgae grow fast with a low lipid content;under stressful conditions,microalgae can synthesize a large amount of lipid but grow slowly.Moreover,the process of stress-induced lipid production in microalgae is usually accompanied by the generation of reactive oxygen species(Reactive Oxygen Species,ROS),which mainly include hydrogen peroxide(H₂O₂),superoxide anion(O₂^·-)and hydroxyl radical(OH)·).There are many studies have shown that ROS play an important role in the growth and lipid production of microalgae,however the production pathway and mechanism of ROS are still unclear.In this study,Chlorella pyrenoidosa was used as the research object to analyze the function of NADPH oxidase in the production of ROS,to investigate the effect of ROS small molecules on the growth and lipid production of C.pyrenoidosa,and to analyze its molecular mechanism.The main research contents and results are as follows:(1)The NADPH oxidase activators Ca²⁺and NADPH as well as the inhibitors EGTA,La Cl₃,DPI and BAPTA were used in order to investigated the role of NADPH oxidase in ROS production.The results showed that the addition of Ca²⁺or NADPH could significantly increase the concentration of intracellular small ROS molecules H₂O₂,O₂^·-and OH·)in C.pyrenoidosa.The concentration of intracellular ROS small molecules was higher under nitrogen and phosphorus deficiency conditions,and after the addition of NADPH oxidase inhibitors(EGTA,La Cl₃,DPI and BAPTA),the intracellular ROS level was significantly reduced,indicating that NADPH oxidase plays an important role in the production of ROS.NADPH oxidase is another important component of ROS generation in microalgae besides mitochondria,chloroplasts and lysosomes.(2)It was found that H₂O₂ can promote the overexpression of C.pyrenoidosa growth-related genes for the first time.C.pyrenoidosa was treated by H₂O₂ with a concentration of 2 m M,74 differentially expressed genes related to DNA replication and ribosome synthesis were found,of which 72 genes were significantly up-regulated.The detection of cell cycle showed that the proportion of C.pyrenoidosa cells in the S phase of the cell cycle increased under the treatment of 2 m M H₂O₂,indicating that the proliferation ability of microalgae increased.These results suggest that 2 m M H₂O₂promotes growth by overexpressing genes involved in cellular DNA replication and RNA translation.The results can provide gene targets for related research on improving algal biomass production through genetic engineering.(3)Under the conditions of nitrogen deficiency and phosphorus deficiency,the levels of intracellular ROS and three ROS small molecules(H₂O₂,O₂^·-and OH·)were investigated,also the correlation between them and lipid content was analyzed.The results showed that the OH·concentration was highly correlated with the lipid content,and the correlation index was as high as 0.938.The Fenton-like reaction was constructed in order to induce OH·specifically and increase the intracellular OH·concentration,it was found that the lipid content was significantly increased at this time,indicating that OH·was involved in the induction of lipid synthesis.(4)The mechanism of OH·regulating lipid synthesis was analyzed via the combined analysis technology of transcriptomics,enzymology and targeted metabolomics.It was found that the acetyl-Co A carboxylase gene of C.pyrenoidosa was significantly up-regulated under the action of OH,and the enzymatic activities of acetyl-Co A carboxylase were significantly increased while the enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase were significantly decreased,which result more carbon enter into fatty acid synthesis pathway and pentose phosphate pathway,thereby providing a large amount of carbon and energy for lipid synthesis.