Transcriptional insights into Chlorella sp. ABC-001: a comparative study of carbon fixation and lipid synthesis under different CO2 conditions

Abstract

Background: Microalgae's low tolerance to high CO₂ concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO₂ content-an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO₂ conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO₂-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO₂) and at various growth phases.

Results: Cells cultivated with 10% CO₂ exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO₂ concentrations and phenotypes, transcriptomic analysis was conducted across different CO₂ conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO₂ conditions, resulting in improved CO₂ fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO₂ conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae.

Conclusions: We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO₂ concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.

Keywords: CO2 fixation; Chlorella; Lipid accumulation; Microalgae; Transcriptomic analysis.

Fig. 1

Cultivation of Chlorella sp. ABC-001 under different CO₂ conditions. a Growth curve of cells cultivated with ambient air and 10% CO₂. The line graph represents cellular density, and the bar graph represents dried cell weight. b The lipid contents of cells as measured after transesterification into FAME. c Changes in nitrate concentration (NO₃⁻) and sulfate concentration (SO₄²⁻). d pH changes. Error bars stand for the standard error calculated from three independent experimental data sets. Asterisks indicate the significant difference between cells cultivated under ambient air and 10% CO₂ determined by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Transcript profiling changes by CO₂ concentration. a The numbers of upregulated and downregulated DEGs in each condition. b Venn diagram representing the number of non-redundant DEGs from different CO₂ concentrations. The left and right Venn diagram each represents upregulated and downregulated DEGs. C1A, C3A, and C3A represent the DEGs comparison of C1 vs A1, C3 vs A3 and C7 vs A7, respectively

Fig. 3

GO analysis of upregulated and downregulated non-redundant DEGs during cultivation under different CO₂ concentrations. The top 7 terms were listed in each analysis based on the cellular growth phase of day 1 (a, d), day 3 (b, e), and day 7 (c, f). The size of the circle represents the gene numbers, and the reference is on the left bottom side of the figure

Fig. 4

Relative expression levels (log₂ fold change) of key enzymes participating in carbon fixation, Calvin cycle, nitrogen uptake, and lipid biosynthesis. The Gene IDs of each enzyme are listed in Additional file 1: Table S3. (GAPDH Glyceraldehyde-3-phosphate dehydrogenase, GPDH glycerol-3-phosphate dehydrogenase, GPAT glycerol-3-phosphate acyltransferase, LPAT lysophosphatidic acid acyltransferase, PAP phosphatidic acid phosphohydrolase, DGAT diacylglycerol acyltransferase, PDAT phospholipid-diacylglycerol acyltransferase, bHLH basic helix-loop-helix, MYB myeloblastosis, bZIP basic leucine zipper, ACC1 Acetyl-CoA carboxylase 1, FAD Fatty acid desaturase)

Fig. 5

Validation of RNA-sequencing data by qRT-PCR. Each data was normalized by the expression level of A1. Error bars stand for the standard error calculated from three independent experimental data sets. Specific gene IDs are listed in Additional file 1: Table S3

Fig. 6

Overall metabolic changes in Chlorella sp. ABC-001 at different CO₂ environments. The color of each enzyme represents either upregulation (red) or downregulation (blue) of genes on day 1. Small boxes stand for the log2 fold changes of representative enzymes of each metabolic reaction. The asterisk indicates adj.p < 0.05. Specific gene IDs are listed in Additional file 1: Table S3. (FeH ferredoxin hydrogenase, SBPase sedoheptulose-1,7-bisphosphate, FBPase fructose 1,6-bisphosphatase, GAPDH glyceraldehyde-3-phosphate dehydrogenase, CA carbonic anhydrase 4, NR nitrate reductase1, NiR nitrite reductase1, METE cobalamin independent methionine synthase, Met methionine, Lipd syn represented by PDAT1, Starch syn represented by SS)