Multi-omics Study On Metabolic Regulation Driven By CO₂ In Biofuel-producing Microalgae

Given the fact that modern society excessively rely on traditional energy,green house effects and energy crisis have received worldwide attention.There is an urgent need for a new energy resource that is renewable and environmental-friendly,which leads to the rapid development of biofuels research.Microalgae consist of extremely diverse unicellular photosynthetic microorganisms that can fix CO₂ and convert solar energy into chemical energy efficiently.They have emerged as alternative feedstock for biofuels production with several advantages,such as high growth rate,high lipid yield and not competing with food crops or forestry for arable land and clean water,Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds and their global regulation upon elevated CO₂ is crucial to promote biomass production,carbon fixation and lipid production via microalgae,which is the main effort and hotspot in microalgae biotechnology.In the present study,Chlorella vulgaris,a microalga with good CO₂ fixation capacity,and Coccomyxa subellipsoidea that is oleaginous and cold-resistant,were thoroughly investigated.Cytomics techniques were employed to evaluate the their variation of physiology and biochemical components,as well as carbon fixation and lipid production performance.The underlying mechanisms of metabolic regulation were further elucidated via metabolomic and transcriptomic analysis.The main results can be summarized as follows:?1?C.vulgaris CS-42 was cultivated phototrophically in two cylindrical photobioreactors with aeration of 5%?v/v?CO₂ or air respectively for 13 d to evaluate the effects of CO₂ supplementation on biomass,CO₂ fixation performance and biochemical content.Significant increases in specific growth rate and total carbon content resulting in a higher CO₂ fixation rate was found with 5% CO₂.The maximal biomass concentration,carbohydrate and fatty acid contents with 5% CO₂ were significantly higher than those with air,while carbohydrate biosynthesis was most affected as compared to other biochemical components.Cytomic analysis revealed a rapid accumulation of neutral lipid in the late growth phase with more lipid bodies visualized by a confocal laser scanning microscope,when nitrate consumption was accelerated with CO₂ supplementation.GC-MS analysis indicated that 5% CO₂ favored the formation of C18:2,which led to a decrease in the degree of lipid unsaturation?DLU?.These results proved that CO₂ supplementation was one of the most efficient methods to significantly prompt the growth of microalgae and increase the C/N ratio in the medium,which in turn regulated the carbon metabolic flux to enhance neutral lipid and fatty acid production in C.vulgaris.?2?By using GC/TOF-MS based approach,metabolite profiles in cells from 0.04% and 5% CO₂ groups sampled at 192,240,288 and 312 h were obtained.178 metabolites were identified and relatively quantified in both cultures.Multivariate statistical analysis was employed to classify the metabolome data and respectively discover 13 and 26 biomarkers for 288 and 312 h in response to CO₂ supplementation,suggesting carbohydrates and amino acids level showed an opposite behavior in CO₂ stress group during the later stage of the cultivation.Pathway analysis showed notable downregulation in metabolites involved in glycolysis and TCA cycle,which led to accumulation of glucose and other sugars.These suppressed pathways together with limited supply of N source at the late stage of cultivation,might weaken amino acid synthesis;Inhibited TCA cycle to some degree boosted lipid accumulation as more acetyl-CoA was funneled towards fatty acid biosynthesis;2 biomarkers associated with ornithine pathway significantly decreased,implicating that nitrogen assimilation and recycling was greatly enhanced to maximize nitrogen utilization.?3?We have explored the physiological variation in C.subellipsoidea C-169 in response to CO₂ level,as well as its capacity of carbon fixation and lipid production.Resultd showed that 2% CO₂ is optimal for the growth of C-169,which significantly enhanced biomass productivity(222 mg L^-1 d^-1),fatty acid content?48.5% dry weight?and productivity(108 mg L^-1 d^-1),and carbon fixation rate(0.431 mg L^-1 d^-1)/ These results suggest that C-169 is a promising candidate for CO₂ biofixation and biofuels production.?4?Transcriptome analysis was performed on C-169 cultured under 0.04% and 2% CO₂ supplementation for 4 day.The sequenced libraries generated reads representing 9409 genes,which account for 96% of the protein coding genes of C-169.The sequencing data was valided by qRT-PCR and deposited in GEO database.1737 differentially expressed genes?DEGs?were identified,with 871 up-regulated and 866 down-regulated.DEGs were annotated by GO term and KEGG orthology.GO enrichment analysis showed that upregulated genes were enriched in ATPase coupled proton transporter,tricarboxylic acid?TCA?cycle,nitrogen compound metabolism;while the downregulated genes were significantly enriched in photosynthesis,including light harvesting,photosystem I and photosystem II.Pathway enrichment analysis suggested that series of pathways were dramatically modulated,including carbon fixation,glycolysis/gluconeogenesis,TCA cycle,pentose phosphate pathway,nitrogen metabolism,oxidative phosphorylation,etc.?5?Detailed pathway analysis was conducted on central metabolic pathways of C-169 according to KEGG database.Results showed that other than enhancing gene expression in the Calvin cycle,C-169 upregulated the expression of phosphoenolpyruvate carboxylase,pyruvate carboxylase and carbamoyl-phosphate synthetase II to enhance the anaplerotic carbon assimilation reactions upon elevated CO₂.Upregulation of ferredoxin and ferredoxin-NADP+ reductase implied that plentiful energy captured through photosynthesis was transferred through ferredoxin to sustain rapid growth and lipid accumulation.Genes involved in the glycolysis,TCA cycle and oxidative phosphorylation were predominantly upregulated presumably to provide abundant intermediates and metabolic energy for anabolism.Coordinated upregulation of nitrogen acquisition and assimilation genes,together with activation of specific carbamoyl-phosphate synthetase and ornithine pathway genes,might help C-169 to maintain carbon/nitrogen balance upon elevated CO₂.Significant downregulation of fatty acid degradation genes,as well as the upregulation of fatty acid synthesis genes at the later stage might contribute to the tremendous lipid accumulation.