Screening And Identification Of Regulatory Genes Involved In Lipid Synthesis And Accumulation In Coccomyxa Subellipsoidea C-169 Under Nitrogen-limitation Induction

The regulation genes involved in intracellular lipid synthesis and accumulation pathway was one of the keys to further improve lipid productivity with genetic engineering strategy and realize the low-cost industrialization of alga-based biodiesel.However,due to the lack of cognition and precise research on the key regulatory genes involved lipid synthesis and accumulation pathway consequently using genetic engineering strategy to enhance algal lipid accumulation lack theoretical guidance.In this study,Coccomyxa subellipsoidea C-169,a novel energy source,was used as a test object.Based on the transcriptomics and metabolomics correlation analysis techniques,the quantitative relationship between lipid accumulation and nitrogen limited concentration-time effect of Coccomyxa subellipsoidea C-169 was first analyzed.Based on this,the related genes regulating the synthesis and accumulation of lipid in the cells of Coccomyxa subellipsoidea C-169 were screened,and further gene overexpression pattern were used to confirm the positive and negative correlation between gene expression pattern and lipid accumulation,lay the theoretical foundation for accurately carry out the genetic engineering of lipid-producing microalgae.The main research results are as follows:1)In the N-replete coupling N-limitation two-stage induction mode,the maximum biomass and lipid content of the cells obtained from the first stage of N-replete basal medium?1.25 g/L NaNO₃?for 168 h was 3.26 g/L and 36%,respectively.the first stage of 3.26 g/L biomass of the Coccomyxa subellipsoidea C-169 was collected and resuspended in fresh,second stage nitrogen-limitation basal medium?0,0.25,0.5,0.75,1.0,1.25 g/L NaNO₃?induced culture for 2 h,4 h,6 h,12h,24 h,36 h and 72 h,respectively.The 0.5 g/L NaNO₃ induced 4 h to obtain the maximum lipid content was 66%and the corresponding biomass was 4.78 g/L.2)The samples of Coccomyxa subellipsoidea C-169 cells cultured in the first stage by two-stage method?3.26 g/L and 36%?were used as the control group,and the samples of Coccomyxa subellipsoidea C-169 cells with the largest lipid accumulation in the second stage?4.78 g/L and 66%?were used as the experimental group for transcriptome analysis to screen the differentially expressed genes.The results showed that 541 genes were significantly changed,513 genes were significantly up-regulated and 28 genes were significantly down-regulated.124 genes were concentrated in the KEGG pathway database,which were related to glycolysis,pentose phosphate pathway,oxidative phosphorylation pathway,glyceride and fatty acid synthesis pathway.Further,phosphoenolpyruvate carboxylase gene?gene4216?and pyruvate dehydrogenase gene?gene899?in the glycolytic pathway and long-chain acyl-CoA synthetase gene?gene8195?,malonyl coenzyme A transferase gene?gene1393?and 3-ketoethyl acyl carrier protein synthase gene?gene9592?in the triglyceride synthesis pathway were identified by real-time quantitative analysis of five key genes in qRT-PCR,found that the expression patterns of these selected genes were consistent with the transcriptome,it was confirmed that the data of differentially expressed genes screened by transcriptome were reliable.3)Similarly,the samples of Coccomyxa subellipsoidea C-169 cells cultured in the first stage by two-stage method?3.26 g/L and 36%?were used as the control group,and the samples of Coccomyxa subellipsoidea C-169 cells with the largest lipid accumulation in the second stage?4.78 g/L and 66%?were used as the experimental group for metabolomics analysis to screen the differentially metabolites.The results showed that 140 metabolites changed significantly,54 metabolites were down-regulated and 86 metabolites were up-regulated.The KEGG pathway database analyzed 24 differential metabolites including fatty acid,phosphatidic acid,phosphatidic acid alcohol,phosphatidyglycerol,diglyceride,phosphatidylserine,phosphatidylethanolamine,phosphatidylcholine are concentrated in the path directly related to lipid accumulation,such as glutathione metabolism,phosphoinositol metabolism and fatty acid synthesis.4)There were 10 correlative pathways between transcriptome differentially expressed genes and metabolome differentially expressed metabolites.The results showed that the expression of glutathione and glucose-6-phosphate was up-regulated in the glycolytic pathway associated with lipid accumulation,the phosphoenolpyruvate carboxylase ppc gene?gene4216?and pyruvate dehydrogenase PDHA gene?gene899?in the glycolytic pathway were also significantly up-regulated,which confirmed that gene4216 and gene899 were the key genes for the regulation of lipid enrichment;the content of oleic acid in the differentially expressed metabolites increased significantly,and the differentially expressed genes that regulate fatty acid synthesis and accumulation such as acetyl-CoA carboxylase gene1159?accC?,gene2002?accA?,gene2481?accB?,malonyl CoA transferase fabD gene?gene1393?,3-oxalyl acyl carrier protein synthetase gene gene9592?fabF?were also up-regulated,which indicates that these genes were important regulators of fatty acid synthesis and accumulation;the increased enrichment of phosphatidylic acid?PA?and DAG were consistent with the expression patterns of fatty acid thioesterase gene gene823?FATA?and long-chain acyl-CoA synthase gene gene2932?ACSL?,which regulated the accumulation of triglyceride synthesis.5)In order to further confirm the role of the selected genes in the regulation of lipid synthesis and accumulation,the pyruvate dehydrogenase gene?gene899?was cloned by gene engineering strategy and transduced into the biological model algae Chlamydomonas reinhardtii for overexpression,the results showed that the biomass of the transgenic Chlamydomonas reinhardtii had no significant change compared with the non-transgenic algal strain,while the lipid content was 59.61%,which was1.84 times higher than that of the non-transgenic Chlamydomonas reinhardtii.In addition,the intracellular fatty acid composition of the transgenic algal strain is mainly composed of C16-C18 fatty acids,making them ideal raw materials for the preparation of high-quality biodiesel.