The influence of exogenous organic carbon assimilation and photoperiod on the carbon and lipid metabolism of Chlamydomonas reinhardtii

Microalgae are a promising platform for the production of renewable fuels and oleochemicals. Despite significant research efforts to understand the mechanisms of algal lipid accumulation, the influence of commercially relevant growth conditions on the lipid metabolism is poorly understood. To characterise the impact of differing organic carbon availabilities and photoperiod on the response of the model alga Chlamydomonas reinhardtii to nitrogen stress, the expression of key genes involved in the central carbon metabolism were monitored over a time-course of nitrogen deprivation. In addition, the growth, PSII integrity, chlorophyll content, triacylglycerol (TAG) content, starch content, and fatty acid composition were characterised. Results indicate that both organic carbon availability and photoperiod regulate the lipid accumulation response of C. reinhardtii. Under mixotrophic conditions, organic carbon uptake is favoured over photosynthesis, transcript abundance of lipid synthesis genes rapidly increase and acetate is funnelled to TAG synthesis. In contrast, autotrophic cultures lacking organic carbon experienced a slower rate of photosynthetic degradation and funnelled the majority of sequestered carbon to starch synthesis. Dark periods induced catabolism of both starch and TAG in autotrophic cultures but TAG alone in mixotrophic cultures. Furthermore, diurnal light enhanced starch synthesis under mixotrophic conditions. Finally, transcript analysis indicated that PGD1, important for the routing of oleic acid to TAG, was reliant on organic carbon availability, resulting in reduced C18:1 fatty acid accumulation in autotrophic cultures

Role and localization of the glyoxylate cycle in the model microalga Chlamydomonas reinhardtii cultivated under day/night cycles

The ability of Chlamydomonas reinhardtii to grow under heterotrophic conditions, without light and solely with an exogeneous organic carbon source (acetate) in its cultivation medium, is one the particular features that have brought this green microalga to be considered as a model organism for the last 50 years, especially for studies on photosynthesis. Assimilation of acetate as an energy source is enabled by the glyoxylate cycle (GC), whose steps are quite similar to the ones of the Krebs cycle but comprises two specific enzymes, namely isocitrate lyase (ICL) and malate synthase (MAS). This cycle is well-known in other model organisms such as the yeast S. cerevisiae and the land plant A. thaliana but has been only poorly studied in Chlamydomonas since its discovery back in 1957, and more particularly when cells are cultivated under alternating light and dark phases, conditions under which it is thought to play a crucial role for the development of the alga. During this project, we started by precisely identifying the isoforms of the enzymes comprised in the GC in addition to their subcellular localisation. It came out that all of them are located inside the peroxisomes with the notable exception of ICL which is located in the cytosol. We then studied the evolution with time during two consecutives day/night cycles (12h/12h) of some of the main central carbon metabolism molecules which are tightly interconnected with the glyoxylate cycle. This was performed with control strains at first and then with a mutant strain deficient for the ICL gene, allowing us to observe that most of these metabolites accumulation profiles are influenced by the cell cycle and division rate, and that the GC is essential for the growth of C. reinhardtii under such alternate light conditions with acetate supplemented in the cultivation medium. Finally, in order to have a better comprehension of the cellular regulation mechanisms linked to these phenotypical observations, we performed a transcriptional analysis of data sampled from the same cultivation conditions using a method named “Surprisal Analysis”. Application of this original method enabled to show that control cells are highly influenced by acetate metabolism during day phase and by replication mechanisms during night phase while mutant cells, which are unable to divide, are markedly influenced by stress-related pathways during both phases but more particularly when light is turned off and they have therefore access to no source of energy to maintain their metabolism

Contribution to the study of mitochondrial metabolism and genome in Chlamydomonas reinhardtii

For the last decade, there has been growing concern about oil reserves depletion. Chlamydomonas reinhardtii, as well as other microalgae, has been investigated with a view to producing biofuel. But current production costs are still too high and impede the commercial implementation of the biofuel from microalgae strategy. Genetic improvement of C. reinhardtii is, thus, an area of immense interest in the worldwide scientific community. During this work, we studied a mutant for the isocitrate lyase (ICL) enzyme. It is the key enzyme of the glyoxylate cycle, which allows Chlamydomonas to use acetate as a carbon source to grow and develop, especially when cultivated in the dark. Identification and characterization of effects of this mutation could, thus, be helpful to better understand the interplays between ICL and other metabolic pathways. This study of the icl mutant highlights modifications of several cellular functions such as respiration, amino acids biosynthesis and stress responses. The second part of this work was devoted to the determination of mitochondrial transcription start site(s) in Chlamydomonas reinhardtii. Although mitochondrial genome transformation can be performed for several years, little is known about the mechanism of the mtDNA transcription in Chlamydomonas and especially about the promoter(s) used. In this work, we establish an almost functional protocol for transcription start site(s) identification and results suggest that multiple promoters might exist, which has never been observed before

RNA-seq data and surprisal analysis of icl mutant and control strain of the green microalga Chlamydomonas reinhardtii during day/night cycles

The data presented in this article are associated to the research article “Surprisal analysis of the transcriptomic response of the green microalga Chlamydomonas to the addition of acetate during day/night cycles” (Willamme et al., 2018) [1]. Here the RNA-seq data of the icl mutant, a null mutant of the isocitrate lyase gene, and its control are summarized and the FPKM values are listed. The data were analysed using surprisal analysis and the genes contributing the strongest to the mutant and wild type phenotype are listed. The raw data are accessible at BioProject PRJNA437393 with SRA accession number SRP136101 (experiments SRX3824204–SRX3824249). The raw data set and expression values used for surprisal analysis are made public to enable critical or extended analyses

Surprisal analysis of the transcriptomic response of the green microalga Chlamydomonas to the addition of acetate during day/night cycles

Our study aims to find gene pathways that depend on acetate assimilation under diurnal conditions in the microalga Chlamydomonas. We compare the transcriptome of two strains, one control and one mutant deficient for the glyoxylate cycle essential in acetate metabolism, cultivated under day/night cycles with acetate. We apply surprisal analysis, an information-theoretic approach, to the RNA-seq data. Carrying out the analysis on groups of dark and light phase samples separately allows identifying constraints and gene pathways that discriminate between mutant and control samples. Carbon metabolism is the most important in the light phase for the control strain while the dark phase is enriched in cell division pathways. The mutant phenotype includes genes pathways of stress response and autophagy in the two phases. Cell division pathways are found in the light phase and catabolic pathways in the dark phase, highlighting a rewiring of the mutant transcriptome in these cyclic cultivation conditions

Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii

Lauersen KJ, Willamme R, Coosemans N, Joris M, Kruse O, Remacle C. Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii. Algal Research. 2016;16:266-274

Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions

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