Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome

Abstract

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Figure 1.

Circular View of the Genomic Landscape of F. solaris JPCC DA0580. Forty-two chromosome pairs are represented with genes (red for positive strand and light red for negative strand) and repeat elements (green for positive strand and light green for negative strand). The chromosome numbers (black) are marked on the outermost circle (dark blue). Within each chromosome pair, the chromosome with higher GC content is defined as chrX (X is the chromosome number) and the other as chrX′. The inner four tracks (blue and orange) of genes and repeats are for the second pair of 42 chromosomes. The innermost circle is a pie chart showing the percentage of each genomic feature in diploid genome coverage.

Figure 2.

Venn Diagrams of Shared/Unique Gene Families of F. solaris JPCC DA0580. Numbers of gene families belonging to each area are indicated. (A) Comparison of whole gene families between F. solaris JPCC DA0580 (fso), T. pseudonana (tps), and P. tricornutum (pti). (B) Comparison of F. solaris-specific gene families with whole gene families of the oleaginous Eustigmatophyte, N. gaditana (nga).

Figure 3.

Monitoring Neutral Lipid Accumulation in F. solaris JPCC DA0580. (A) Differential interference microscopy (left) and fluorescence microscopy (right) of F. solaris cells stained with BODIPY 505/515. Red and green represent chloroplast and oil bodies. (B) Growth (closed circles) and nitrate concentrations in the culture medium (open circles). Data are means of triplicate experiments ± sd. (C) Neutral lipid content (closed triangles) and carbohydrate content (open triangles).

Figure 4.

Differential Gene Expression during Oil Accumulation. The 20 genes with the highest RPKM values in the major pathways (i.e., glycolysis, gluconeogenesis, TCA cycle, nitrogen metabolism, fatty acid degradation, glycerolipid biosynthesis, and Calvin-Benson cycle) are shown at each time point (48-, 96-, and 144-h culture). Transcription profiles are depicted as heat maps, where yellow and blue indicate up- and downregulation, respectively, as shown in the scale bar (log₂ ratio). GPAT (g9180) in glycerolipid synthesis ranked off from the list at 144 h because of a number of genes in fatty acid degradation ranked in it, although GPAT remained in high RPKM.

Figure 5.

Expression of Genes Encoding Fatty Acid and TAG Biosynthesis Pathway Components in F. solaris JPCC DA0580. The 96-h (A) and 144-h (B) culture. Each box represents an isoform. Box sizes and numbers represent magnitudes of RPKM values [3: (8)∼16, 4: ∼32, 5: ∼64, 6: ∼128, 7: ∼256, 8: ∼512, 9: ∼1024, 10: 1024∼]. Red, black, and green boxes represent log₂ fold changes in RPKM values (96 h/48 h or 144 h/48 h) >1, ≤−1 to 1, and <−1, respectively. Enzymes not predicted to localize in subcellular compartments are indicated by question marks. Homoeologous gene pairs are surrounded by dashed boxes.

Figure 6.

Expression of Genes Encoding Fatty Acid Degradation Pathway Components in F. solaris JPCC DA0580. The 96-h (A) and 144-h (B) culture. Each box represents an isoform. Box sizes and numbers represent magnitudes of RPKM values [3: (8)∼16, 4: ∼32, 5: ∼64, 6: ∼128, 7: ∼256, 8: ∼512, 9: ∼1024, 10: 1024∼]. Red, black, and green boxes represent log₂ fold changes in RPKM values (96 h/48 h or 144 h/48 h) >1, ≤−1 to 1, and <−1, respectively. The enzymes that are not predicted to localize in the subcellular compartments are marked by question marks. The homoeologous gene pairs are surrounded by dashed boxes.

Figure 7.

Proposed Metabolic Scheme for TAG Accumulation in F. solaris JPCC DA0580. Highly upregulated pathways are depicted by red arrows. They include pathways for FA and glycerolipid biosyntheses in the chloroplast. TAG accumulation is supported by the upregulation of parallel pathways, i.e., neosynthesis by the Kennedy pathway and conversion of ER phosphoglycerolipids combined with conversion of chloroplast glycerolipids by an unknown mechanism (?). Catabolism of acyl-CoAs is also upregulated in the mitochondrion.