Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols

Abstract

Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

Fig 1. 18S rRNA phylogenetic tree for marine and freshwater green algae and a marine diatom outlier.

The asterisks indicates organisms with available genomes in the NCBI database. The representative full-length 18S rRNA sequences were aligned with MUSCLE in MEGA 6.

Fig 2. Lipid accumulation in Chlorella strain C596.

(a) Time-course of a batch experiment culminating in N and P co-limitation (N:P = 13:1): nitrate+nitrite (black, solid), phosphate (black, dashed), cells (blue), TAG (red, solid), and total lipids (red, dashed) concentrations; error bars represent the 95% confidence interval. (b-g) Fluorescence and phase contrast microscopy of strain C596 cells grown in batch culture culminating in P limitation (N:P = 30:1). Samples were withdrawn during the exponential phase (b-d) and four days after onset of stationary (e-g) growth phase. The fluorescence microscopy reveals the chloroplast (autoflourescence, bright white) and Nile red stained lipids (in c and f; grey). The red arrows and brackets indicate intercellular TAG locations. The white arrows highlight the chloroplast.

Fig 3. Specific growth rates of Chlorella strain C596.

Growth rates (d^-1, based on cell density over time) are shown for (a) different salinities and (b) various carbon substrates. White bars indicate photosynthetic growth with a PPFD of 80 μmol photons m⁻² s⁻¹; gray bars indicate grown in the dark plus a carbon source at a final concentration of 20 mmol L⁻¹; the black bar indicates growth in the dark. Values are the means of triplicate biological replicates (n = 3); error bars represent ±1 standard error (SE).

Fig 4. Differentially expressed genes between the 182 h versus 109 h samples broken into KEGG categories.

Genes are considered differentially expressed when the edgeR p-value < 0.05 and the fold-ratio > 2 or < 0.5. The assignment of a transcript to a category is based upon the KEGG annotation of orthologs in C. variabilis NC64A. The axis of the dot plot displays the ratio of 182/109 h on a log scale. The numbers in the category column represent the number of genes down-regulated (-), up-regulated (+), and the difference between these (Δ). The color shading is based on the Δ (positive, red; negative, blue).

Fig 5. Detection and monitoring of the TAG biosynthesis pathway in Chlorella strain C596.

(a) The final steps of the TAG biosynthesis pathway. The symbols indicate whether one or more transcripts encode the enzyme at that step is present for Chlorella strain C596 (circle), C. variabilis NC64A (square), or C. reinhardtii (triangle). Grey shading indicates presence. Lined shading for the glycerol-3-phosphate acyltransferase indicates a bacterial-type homolog. Lined shading for the diacylglycerol acyltransferase indicates a secondary homolog in C. reinhardtii. (b) Heatmaps of the cyclophillin-normalized values for the putative transcripts for the TAG accumulation pathway and galactoglycerolipid lipase in samples taken during exponential growth (89 and 109 h) and during lipid accumulation (182 h). The intensity of the shading represents cyclophillin normalized values ranging from 0 to 1.8; note, one transcript, c10691_g3, falls outside of this range and the appropriate box is labeled. The * symbol represents those transcripts for which RT-qPCR primers were designed. The † symbol represents the transcript whose ratio could not be calculated because the transcript was not detected in the 109 h sample. (c) Comparison of the RNA-seq (top row) and RT-qPCR results also normalized to cyclophillin (bottom row) for the predicted DAGAT (left column) and galactoglycerolipid lipase (right column) for two biological duplicates (a and b). The error bars indicate the 95% confidence interval of the technical replicates run for the RT-qPCR.