Regulatory Mechanism Of Cellular Reactive Oxygen Species On Lipid Biosynthesis In Chlorella Pyrenoidosa Under Stress Conditions

In recent years,renewable and sustainable energy resources have garnered much attention due to running out of fossil-based fuels.With higher photosynthetic conversion efficiency,faster growth rate and the ability to store and secrete energy-rich carbohydrates,microalgae can synthesize many high-value compounds,such as starch and lipid.It is known that starch can be used to produce bioethanol while lipid can be used to produce biodiesel.In addition,microalgae have more C₁₆-C₁₈ chain length fatty acids which are good raw materials for biodiesel production.An effective way to increase the content of lipid is applying stress in the cultivation process.The commonly used stress methods are nitrogen starvation,phosphorus starvation,high light,and high salinity.It is found that an increase in intracellular lipid content is often accompanied by an increase in reactive oxygen species?ROS?accumulation in microalgae cells suffering from these stresses.Although many studies have reported this phenomenon,the interaction mechanism has not yet been dealt with.In this work,the physiological and biochemical changes of cells were first explored through four stress conditions:nitrogen starvation,phosphorus starvation,high light and high salinity.Although the cell growth of Chlorella pyrenoidosa?C.pyrenoidosa?was hindered,the lipid accumulation was improved under all stress conditions.Specifically,under the nitrogen starvation condition,the cell growth of C.pyrenoidosa was severely inhibited,with the percentage of cells in the S-phase being only 4.19%,and the cells became smaller and possessed more complicated intracellular structure.Importantly,not only the intracellular lipid content is increased under nitrogen starvation conditions,but also the combustion performance,oxidation stability and low temperature fluidity of the lipid are improved.In addition,it was found that starch was preferentially synthesized by cells under stress.With the prolongation of stress time,starch degraded gradually,and the degraded carbon skeleton was used to synthesize lipid.Secondly,the changes in intracellular oxidative stress and antioxidant system were investigated,and the correlation between total ROS,H₂O₂,O₂^·-,OH^?concentrations and its corresponding lipid content was analyzed through Spearman correlation analysis.The high coefficient of 0.901 was obtained by plotting the OH^?concentration against the corresponding lipid content.Furthermore,with the treatment of 500?mol/L H₂O₂+13?mol/L FeCl₃,a significant increase in cellular OH^?concentration was obtained,while the contents of H₂O₂ and O₂^?-have no significant change.Under such condition,the cellular lipid content was significantly increased,confirming that OH^?can mediate the biosynthesis of cellular lipid.The transcriptome sequencing of C.pyrenoidosa was carried out to confirm that OH^?can mediate the lipid biosynthesis at the gene expression level.A total of 47.74 Gb clean Data were obtained from six groups of samples,and the Q₃₀ base percentage of each sample was more than 95.10%.The filtered Clean Reads were compared with the reference genome of C.pyrenoidosa,and the comparison efficiency ranged from 79.84%to 84.57%.Functional annotation was performed based on the sequenced genes,and10138 gene function annotations were completed,and 1647 unannotated genes were discovered.In order to reveal the changes of gene expression in key intracellular metabolic pathways,differential expression gene analysis was performed.A total of 2104differentially expressed genes were obtained,of which 1808 genes were successfully functionally annotated.It was found that the genes involved in intracellular fatty acid and triglyceride synthesis pathway were significantly up-regulated,and the results of transcriptome sequencing were verified by fluorescence quantitative PCR assay.These results demonstrated that OH^?can mediate the lipid biosynthesis at the genetic level.In addition,the synthesize starch and sucrose increased,while the metabolism of intracellular pigments was significantly reduced under OH^?stress.The results indicate that the reallocation of cellular carbon and ATP from biological functions such as photosynthesis to the synthesis of energy storage molecules?such as starch and lipid?,which enable algal cells to survive under OH^?stress.These results reveal the relationship among stress,reactive oxygen species and lipid for the first time.The stressful conditions can trigger OH^?of ROS,thus increasing lipid biosynthesis.In addition,the metabolism of starch is very important for the lipid biosynthesis,and the degradation of starch can promote the biosynthesis of lipid.This study reveals the regulation mechanism between stress and lipid production,which is of great significance for the production of microalgae biofuel.