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The coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life-cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P-starvation led to an accumulation of many generic and especially N-rich metabolites, including lipids, osmolytes, and pigments. This suggests that P-starvation primarily arrests cell-cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de-epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P-starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N-starvation resulted in a decrease of most central metabolites, also P-containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P-starvation than with N-starvation.

Background Oleaginous microalgae are promising production hosts for the sustainable generation of lipid-based bioproducts and as bioenergy carriers such as biodiesel. Transcriptomics of the lipid accumulation phase, triggered efficiently by nitrogen starvation, is a valuable approach for the identification of gene targets for metabolic engineering. Results An explorative analysis of the detailed transcriptional response to different stages of nitrogen availability was performed in the oleaginous green alga Monoraphidium neglectum. Transcript data were correlated with metabolic data for cellular contents of starch and of different lipid fractions. A pronounced transcriptional down-regulation of photosynthesis became apparent in response to nitrogen starvation, whereas glucose catabolism was found to be up-regulated. An in-depth reconstruction and analysis of the pathways for glycerolipid, central carbon, and starch metabolism revealed that distinct transcriptional changes were generally found only for specific steps within a metabolic pathway. In addition to pathway analyses, the transcript data were also used to refine the current genome annotation. The transcriptome data were integrated into a database and complemented with data for other microalgae which were also subjected to nitrogen starvation. It is available at https://tdbmn.cebitec.uni-bielefeld.de . Conclusions Based on the transcriptional responses to different stages of nitrogen availability, a model for triacylglycerol and lipid hyperaccumulation is proposed, which involves transcriptional induction of thioesterases, differential regulation of lipases, and a re-routing of the central carbon metabolism. Over-expression of distinct thioesterases was identified to be a potential strategy to increase the oleaginous phenotype of M. neglectum, and furthermore specific lipases were identified as potential targets for future metabolic engineering approaches.

To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches. The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3' rapid amplification of cDNA ends (3' RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of Chlamydomonas reinhardtii.