Proteomic analyses bring new insights into the effect of a dark stress on lipid biosynthesis in Phaeodactylum tricornutum

Abstract

Microalgae biosynthesize high amount of lipids and show high potential for renewable biodiesel production. However, the production cost of microalgae-derived biodiesel hampers large-scale biodiesel commercialization and new strategies for increasing lipid production efficiency from algae are urgently needed. Here we submitted the marine algae Phaeodactylum tricornutum to a 4-day dark stress, a condition increasing by 2.3-fold the total lipid cell quotas, and studied the cellular mechanisms leading to lipid accumulation using a combination of physiological, proteomic (iTRAQ) and genomic (qRT-PCR) approaches. Our results show that the expression of proteins in the biochemical pathways of glycolysis and the synthesis of fatty acids were induced in the dark, potentially using excess carbon and nitrogen produced from protein breakdown. Treatment of algae in the dark, which increased algal lipid cell quotas at low cost, combined with optimal growth treatment could help optimizing biodiesel production.

Figure 1. Total lipids, carbohydrates, and proteins as well as key metabolites of Phaeodactylum tricornutum.

(A) Neutral lipid and chlorophyll fluorescence. Neutral lipid bodies were stained into yellow (a,b), red chlorophyll fluorescence observed in bright field (c,d), P. tricornutum grown in the light (a,c), P. tricornutum exposed to a 4-day dark stress (b,d). (B) Proportion of the algal dry weight incorporated in proteins, carbohydrates, lipids, and other minor compounds in P. tricornutum exposed for 4 days in the light (Con) or in the dark (dark). (C) Relative fatty acid composition (in percentage of total cellular fatty acids) of P. tricornutum exposed to optimal light (white bars, Con) and to a 4-day dark stress (black bars, Dark). (D) Cell content of glucose (μmol/10⁶ cells), pyruvate (μmol/ml 10⁻⁶), and acetyl-CoA (μmol/10⁵ cells) in P. tricornutum exposed for 4 d in the light (Con) or in the dark (Dark). Asterisks indicate significant according to independent Student’s t-tests (*P < 0.05, **P < 0.01). Values are mean ± SEM (n = 4).

Figure 2. Classification of the differentially expressed proteins based on functional enrichment and KEGG pathway analysis.

(A) The protein molecular functions, (B) the protein-associated biological processes, (C) the protein class, (D) and the KEGG biochemical pathways.

Figure 3. The transcription of key genes involved in Phaeodactylum tricornutum exposed for 4 days in the light (Con) or in the dark (dark).

(A) Photosynthesis, (B) glycolysis, (C) fatty acid biosynthesis or oxidation. The transcription level of all genes was normalized to that of a housekeeping gene, actin rRNA. Asterisks indicate significant differences between both light treatments according to independent Student’s t-test (*P < 0.05, **P < 0.01). Values are mean ± SEM (n = 4).

Figure 4. Integrated cell mechanisms leading to enhanced lipid production in the dark.

(A) Metabolic fluxes of intermediate metabolites related to intermediate C and N metabolic pathways. The flux line width represents the relative magnitude of the flux. C:N ratio balance represents the control, C:N ratio imbalance represents the darkness treatment group. (B) Conceptual scheme of the dark-induced perturbation of fatty acid synthesis based on iTRAQ results.