Whole Genome Re-Sequencing Identifies a Quantitative Trait Locus Repressing Carbon Reserve Accumulation during Optimal Growth in Chlamydomonas reinhardtii

Abstract

Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named constitutive oil accumulator 1 (coa1). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth.

Figure 1. The constitutive accumulator 1 (coa1) mutant of Chlamydomonas reinhardtii over-accumulates oil during optimal growth in shake-flask cultures.

(A) Triacylglycerol content as quantified by thin layer chromatography (TLC). Data are means of five biological replicates with 95% confidence intervals shown. Stars denote significant increase (Student’s t-test; p < 0.05). (B) Oil content in WT and coa1 (6D8) mutant strains after nitrogen depletion for 3 days. Data are means of three biological replicates and two technical replicates, with 95% confidence intervals shown. Stars denote significant increase (Student’s t-test; p < 0.05). (C,D) Images of Bodipy-stained cells of WT and the mutant coa1. Scale bars = 50 μm. (E,F) Magnification of lipid droplets stained by Nile red. Scale bars = 5 μm. Cells were cultivated in shake flasks in standard TAP medium at a light density of 100 μmol photons m⁻² s⁻¹. Because the coa1 mutant forms cell clusters, biochemical quantification is made based on total cellular volume instead of cell numbers. It is worth noting that for non-aggregated cells of Chlamydomonas, 1 mm⁻³ cellular volumes are equivalent to ~5 million cells.

Figure 2. The coa1 mutant accumulates twice more starch than WT during optimal growth in shake-flask cultures.

(A) Starch content (B) Chlorophyll content. Data are means of six replicates with 95% confidence intervals shown. Stars denote significant increase (Student’s t-test; p < 0.05). Cells were cultivated in shake flasks in standard TAP medium at a light density of 100 μmol photons m⁻² s⁻¹.

Figure 3. Growth characteristics of the coa1 mutant and WT.

(A) Population of WT and coa1 mutant cells was monitored using a cell counter. The mutant cells appear much bigger (~9–10 μm) than WT cells (~5–6 μm) under the same cultivation conditions. (B) Clusters of the coa1 mutant cells were observed under light microscope. Bars = 10 μm. (C) Cell growth for WT and coa1 mutant cells were monitored based either on cell number (solid lines) or on cellular volume (dashed lines). Cells were grown in shake flasks in standard TAP medium at a light density of 100 μmol photons m⁻² s⁻¹. Error bars represent standard deviation based on three biological replicates. Abbreviations: TAP, Tris-Acetate Phosphate.

Figure 4. The coa1 phenotype is positively linked to higher light intensity in photobioreactors operated as turbidostat.

(A) TAG content in cells cultivated under increasing light irradiance. (B) Starch content in cells cultivated under increasing light irradiance. (C) Chlorophyll content in cells cultivated under increasing light irradiance. It is important to note that 40 μmol photons m⁻² s⁻¹ in the PBR set-up being approximately equivalent to 160 μmol photons m⁻² s⁻¹ in the flask batch set-up, roughly a 4-fold change. Data are means of three replicates, and error bars denote 95% confidence intervals. Cells were cultivated in PBRs under strict photoautotrophic conditions maintaining a constant OD_{880 nm} = 0.4 (eq. = 2 million cells mL⁻¹ for WT).

Figure 5. Alterations in the ratio of chlorophyll a versus chlorophyll b (Chl a/b).

(A) Difference in Chl a/b ratio and its evolution in response to increasing light intensity. (B) The chlorophyll cycle and the requirement for a chlorophyll b reductase. Data are means of three biological replicates with two technical replicates each. Error bars represent standard deviations. Chl: chlorophyll.

Figure 6. Genetic analyses confirm a single insertion in the coa1 genome.

(A) DNA blotting of digested genomic DNA of WT and coa1 mutant with three different enzymes and then hybridized with the AphVIII probe. (B) Oil content for the progenies issued from tetrad analyses of coa1 and CC124. (C) Whole genome re-sequencing identifies >200 kb deletion in chromosome 14 between coordinates 69,266 and 317,163. T: tetrad.