Project description:The oleaginous green microalga Neochloris oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation. In order to steer the metabolism towards starch or lipids only, it is important to understand the mechanisms behind it. In this study physiological changes and gene expression upon nitrogen starvation were analysed in controlled flat-panel photobioreactors over both short- and long-term time-scales. Starch accumulation is transient and occurs rapidly within 24 hrs upon starvation, while lipid accumulation lasts longer and reaches a maximum after 4 days. The major fraction of accumulated lipids is composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and is characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated and saturated fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that genes related to starch biosynthesis and degradation show different temporal expression dynamics compared to those of lipid biosynthesis. An immediate rapid increase in starch synthesis gene expression is followed by an increase in starch degradation and decrease in starch synthesis gene expression. In contrast, increased gene expression for fatty acid and TAG synthesis is initiated later and occurs more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of mono-unsaturated and saturated fatty acids. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in N. oleoabundans as well. Promising candidates for future metabolic engineering are a DYRKP homolog that in Chlamydomonas acts as a negative regulator of carbon storage and photosynthetic efficiency under N-starvation, and two bZIP-type regulators that have been implicated to control several steps in microalgal TAG synthesis. Our data for the first time show the temporal dynamics of storage compound accumulation and transcriptional changes on a short- and long-term time-scale during nitrogen starvation in N. oleoabundans, and increases insight into the genetic foundation for starch and lipid metabolism in this microalga. This information and the identified target genes can now be used for metabolic engineering strategies towards tailored N. oleoabundans strains for industrial applications with increased lipid production and altered fatty acid composition.
Project description:In this study, we characterized the fatty acid production in Neochloris aquatica at transcriptomics and biochemical levels under limiting, normal, and excess nitrate concentrations in different growth phases. At the stationary phase, N. aquatica mainly produced saturated fatty acids such as stearic acid under the limiting nitrate concentration, which is suitable for biodiesel production. However, it produced polyunsaturated fatty acids such as α-linolenic acid under the excess nitrate concentration, which has nutritional values as food supplements. In addition, RNA-seq was employed to identify genes and pathways that were being affected in N. aquatica for three growth phases in the presence of the different nitrate amounts. Genes that are responsible for the production of saturated fatty acids were upregulated in the cells grown under a limiting nitrogen amount while genes that are responsible for the production of polyunsaturated fatty acid were upregulated in the cells grown under excess nitrogen amount. Further analysis showed more genes differentially expressed (DEGs) at the loga- rithmic phase in all conditions while a relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase under limiting and excess nitrogen. Our results provide a foundation for identifying developmentally important genes and understanding the biological processes in the different growth phases of the N. aquatica in terms of biomass and lipid production.
Project description:Ettlia oleoabundans is a triacylglyceride-producing chlorophyte. We investigated gene expression and lipid changes after 6 days under varying concentrations of nitrate and NaCl.
