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Towards improving biofuel production - Oil synthesis and accumulation pathways in promising oleaginous microalgae

Final Report Summary - ALGAEOILSYNTH (Towards improving biofuel production - Oil synthesis and accumulation pathways in promising oleaginous microalgae.)

The AlgaeOilSynth project aimed at deciphering, through cellular, molecular and genetic analyses how oleaginous photosynthetic microalgae synthesize and accumulate oil in their cells. Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants. Importantly, the culturing methods of microalgae generally do not compete with agricultural food production, which of high importance in the context of current unfavorable climate changes and growing human population. The key feature of oleaginous microalgae is that under environmental stress conditions, they produce substantial amounts of lipids (20-50 % of their dry weight), mainly in the form of triacylglycerols (TAGs), which are valuable fuel precursors. Therefore, AlgaeOilSynth project was designed to meet the strong need of mechanistic studies on lipid metabolism in microalgae oriented towards developing of new avenues of genetic engineering of a direct use for boosting global biofuel production.
Originally, two model organisms were chosen for realization of this aim – Nannochloropsis oceanica (unicellular green algae) and Phaeodactylum tricornutum (diatom). However, this original choice had to be modified as an intense experimental exploration of P. tricornutum as a potential biofuel source in the last 3 years took place by diverse research groups. This resulted in a high number of reports, the results of which partially overlap with the goals of the AlgaeOilSynth project. Finally, the scientific focus was therefore shifted towards detailed characterization of N. oceanica and led for realization of the following objectives: (i) spatio-temporal analysis of lipid accumulation at cellular level in response to nutrient stress; (ii) identification of the most promising genes governing TAG synthesis; and (iii) their functional characterization in yeast and higher plants.
Among many microalgae considered for biofuel production N. oceanica is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). Remarkably, the genome of N. oceanica, encodes a total of 13 putative diacylglycerol acyltrasferase (DGAT)-encoding genes, which are the enzymes catalyzing the committed step in TAG synthesis. Thus far, a biological rationale for such a large DGAT gene family in N. oceanica is not clear. However, as most of organisms contains much fewer DGAT-encoding genes the overall hypothesis underlying the AlgaeOilSynth project was that the expansion of this specific gene family contributes to the extraordinary capability of N. oceanica to accumulate oil to high amounts and perhaps using more advanced mechanism to regulate this process, when compared to other microalgae strains and oleaginous higher plants.
The results produced during realization of AlgaeOilSynth project led to achievement of its objectives and are summarized below. (i) At cellular level the nutrient stress (nitrogen starvation) of N. oceanica cultures resulted in progressive accumulation of oil in the form of numerous lipid droplets. These organelles were localized in the cytoplasm and after 72 hours occupied most of the cell volume. Concomitantly, lipid accumulation in N. oceacnica cells was accompanied by shrinkage of chloroplasts and reduction of cellular membrane system. Finally, cessation of cell divisions led to significant reduction of growth rates in nitrogen-starved N. oceanica cultures, when compared to cultures growing in optimal conditions; (ii) To match the best candidate DGAT genes directly involved in TAG synthesis in response to nutrient stress the expression profiles of all the 13 DGAT genes encoded by N. oceanica genome were compared between nutrient stress and optimal conditions of growth. Based on the observed expression patterns, the 13 analyzed DGAT genes were categorized into two groups. The first group includes seven DGAT genes, which were unaffected by N deprivation, thus likely are not involved in stress-induced oil accumulation. The second group consists of the remaining six DGAT-encoding genes (NoDGTT1-NoDGTT6) with increased transcript levels following N deprivation. Such expression patterns strongly implies their involvement in massive TAG synthesis in N-starved N. oceanica cells. Differences between the two conditions were particularly strong for one of the DGAT-encoding genes - NoDGTT5, which showed a 20-fold increase at its maximum of expression; (iii) Based on their high expression response, NoDGTT1-NoDGTT6 genes were chosen to test their possible role in TAG biosynthesis and were expressed in a TAG synthesis-deficient mutant of yeast. From all the analyzed DGAT-encoding genes yeast mutant expressing NoDGTT5 accumulated the highest amounts of TAGs. Thus, this DGAT was then selected for further functional characterization. Its over-expression in N. oceanica cells growing in optimal conditions resulted in levels of oil normally observed only after nutrient stress. However, reduced growth rates were observed for these cultures. Constitutive expression of NoDGTT5 in Arabidopsis thaliana plants was accompanied by increased TAG content in seeds and leaves as well. Furthermore, NoDGTT5 was able to successfully rescue the oil deficient Arabidopsis tag1-1 mutant, by restoring the TAG content in seeds comparable to wild type plants.
In summary, AlgaeOilSynth contributed for novel relevant data on lipid synthesis in oleaginous microalgae, which are of direct use for design of new strategies oriented towards biotechnological improvement of oil producing strains. This project revealed that among all the DGATs encoded by N. oceanica genome NoDGTT5 is the most promising gene for the engineering of TAG synthesis in diverse host organisms and it can be used as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production. Beside the experimental aspect, the AlgaeOilSynth project let the applicant gain valuable insights to the state of the art problems of plant research and promoted his scientific maturation by direct interaction with well acknowledged, highly reputed researchers in the field of plant lipids, high impact publications and active participation in cyclic meetings of plant lipid community.
Taking into account the transfer of knowledge between outgoing and return host institutes, the record of publications, the milestone and deliverable produced, the overall and specific objectives of AlgaeOilSynth project were fully achieved.