Global transcriptomic changes following N-deprivation in Nannochloropsis oceanica IMET1: Illumina sequencing
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ABSTRACT:
ORGANISM(S): Nannochloropsis oceanica
PROVIDER: GSE42508 | GEO | 2014/05/14
SECONDARY ACCESSION(S): PRJNA182180
REPOSITORIES: GEO
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Project description:Limited systems-level understanding of oil synthesis in wild oleaginous algae has hindered the development of microalgal feedstock. Nannochloropsis is a small unicellular microalgae widely distributed in oceans and fresh water. In many large-scale and pilot-scale outdoor cultivation facilities, Nannochloropsis strains have been found to be capable of robust growth when supplied with flue gases, naturally accumulating large quantities of oils in a stationary phase, and exhibiting resistance to environmental contaminants. The rich genomic resources, compact genomes, resistance to foreign DNA invasion, wide ecological adaptation, large collections of natural strains and the demonstrated ability to grow on a large scale suggested Nannochloropsis can serve as research models and platform strains for economical and scalable photosynthetic production of fuels and chemicals. To untangle the intricate genome-wide networks underlying the robust biomass accumulation and oil production in Nannochloropsis, we applied high-throughput mRNA-sequencing and reconstructed the structure and dynamics of the genome-wide functional network underlying robust microalgal triacylglycerol (TAG) production in Nannochloropsis oceanica, by tracking the genome-wide, single-base-resolutiontranscript change for the complete time-courses of nitrogen-depletion-induced TAG synthesis. Nannochloropsis oceanica IMET1 cells were grown in liquid cultures under continuous light (approximately 50 M-BM-5mol photons m-2 s-1) at 25M-bM-^DM-^C and aerated by bubbling with a mixture of 1.5% CO2 in air. Mid-logarithmic phase algal cells were collected and washed three times with axenic seawater. Equal numbers of cells were re-inoculated in nitrogen replete medium (Control condition or C, i.e. N+) and nitrogen deprived medium (N deficiency or N, i.e. N-) with 50M-BM-5mol m-2 s-1 light intensity, respectively. Cell aliquots were collected for RNA isolation after being transferred to the designated conditions for 3h, 4h, 6h, 12h, 24h and 48h. Three biological replicates of algal cultures were established under each of the above M-bM-^@M-^\CM-bM-^@M-^] (i.e. N+) and M-bM-^@M-^\NM-bM-^@M-^] (i.e. N-) conditions, respectively. In total, 36 samples collected at six time points (3h,4h,6h,12h,24h and 48h) were used for mRNA-Seq library preparation and then submitted to Illumina HiSeq 2000 for sequencing.
2014-05-14 | E-GEOD-42508 | biostudies-arrayexpress
Project description:Limited systems-level understanding of CO2 concentrating mechanism (CCM) and metabolic adaption in response to different CO2-level in wild oleaginous algae has hindered the development of microalgal feedstock and the knowledge of its role in global warming and oceanic acidification. Nannochloropsis are a group of small unicellular microalgae widely distributed in oceans and fresh water, which implies that it plays a crucial role in biogeochemical cycles impinged on global climate change. In addition, Nannochloropsis has been used for flue gas fixation in many large-scale and pilot-scale outdoor cultivation facilities for photosynthetic production of fuels and chemicals. To untangle the intricate genome-wide networks underlying CCM and metabolic adjustment under different CO2 concentrations in Nannochloropsis, we applied high-throughput mRNA-sequencing and reconstructed the structure and dynamics of the genome-wide functional network underlying robust microalgal CCM and in Nannochloropsis oceanica, by tracking the genome-wide, single-base-resolution transcript change for the complete time-courses of different CO2 concentrations.
2019-07-16 | GSE55861 | GEO
Project description:Oleaginous microalgae are considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields are too low to allow for an economically feasible production process. Lipid yields could be enhanced by improving microalgal strains through genetic engineering. Strain improvement strategies for the industrially relevant genus Nannochloropsis have met limited success because most genes of this genus lack a functional annotation, hindering our understanding of lipid metabolism and its regulation. To gain fundamental insights and to provide targets for genetic engineering of lipid metabolism, the aim of this study was to discover novel genes that are associated with higher neutral lipid (NL) content in Nannochloropsis oceanica. Therefore, we constructed a random gene knockout (KO) insertional mutagenesis library of N. oceanica, and we screened it by five rounds of fluorescence-activated cell sorting to select high lipid mutant (HLM) strains. Several strains showed increased NL contents compared to the wild type under favorable growth conditions. By using an adapted cassette PCR strategy involving the type IIS restriction endonuclease MmeI, we traced the responsible genetic KO of the five most promising mutant strains. One particularly promising mutant strain (HLM23) was disrupted in gene NO06G03670, which encodes a putative APETALA2-like transcription factor. HLM23 was not affected in growth rate, had increase d photosynthetic performance and a NL content of 30% dry cell weight^(-1), a 40% increase compared to the wild type. RNA sequencing revealed a transcriptional upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin–Benson–Bassham cycle in this mutant.
2021-05-13 | GSE167058 | GEO
Project description:Microalgal lipid, a feasible substrate for biofuel, is typically accumulated during the stationary growth phase. Generating strains which trigger lipogenesis from the exponential growth phase will enhance lipid productivity, reduce cost of biofuel production. We characterized a lipid-rich microalgal mutant, Dunaliella tertiolecta, which exhibited a six-fold enhancement of neutral lipids production in the exponential growth phase with marginal compromise on growth (4%). Using transcriptomics and metabolomics, regulatory mechanisms of the mutant were uncovered.
2016-10-24 | GSE82121 | GEO
Project description:Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high value lipid products. First success in applying reverse genetics makes Nannochloropsis species attractive models to investigate the cell and molecular biology and biochemistry of this fascinating organism group. (Principle findings) Here we present the assembly of the 28.7 Mb genome of Nannochloropsis oceanica CCMP1779. RNA sequencing data from N-replete and N-depleted growth conditions support a total of 11,973 genes, which in addition to automatic annotation were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors and 109 transcriptional regulators were annotated. In addition, we provide protocols for the transformation of the sequenced strain. (Significance) The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols provides a blueprint for future detailed gene functional analysis and phylogenetic comparison of Nannochloropsis species by a growing academic community focused on this genus. one sample each of nitrogen-replete and nitrogen-depleted conditions
2012-04-04 | E-GEOD-36959 | biostudies-arrayexpress
Project description:Purpose: The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells. However the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. Methods: Nannochloropsis cells (50 ml per sample) were harvested for RNA isolation during nitrogen deprivation (N-) and after nitrogen resupply (NR) at the following time points: 6, 12, 24, 48 and 72 h. For each time point two biological replicates were harvested. RNA was extracted using the E.Z.N.A® Plant RNA kit (OMEGA) according to the manufacturer’s instructions. RNA quality was verified using the Bioanalyzer (Agilent). Single-end, 50 bp nucleotide sequences were acquired for each sample using an Illumina HiSeq 2500 (MSU-Research Technology Support Facility). RNA-Seq reads were trimmed using Trimmomatic (v0.32) with removal of leading and trailing low quality bases (below quality score 10 with a 4-base wide sliding window) as well as TruSeq Single End adaptors. The resulting reads were aligned to the N. oceanica CCMP1779 genome assembly (GSE36959) using STAR (2.3.0e) (Dobin et al., 2013) by only allowing unique mapping, a maximum of 4 mismatches per pair and a maximum intron length of 10,000 bp. The HTSeq (0.6.1) was used to generate a count table for all genes using the exon as the feature type as well as the intersect-nonempty mode so that only reads that were completely aligned to two genes are considered ambiguous. HTSeq count tables were used for differential expression analysis using DEseq2. Differential gene expression was examined using DESeq2. A generalized linear model was fit, and we tested on condition (deprivation or recovery) and the time:condition interaction with log2-fold changes greater than 1. Significant differentially expressed genes with p-values less than 0.01 were identified after Benjamini-Hochberg (BH) multiple testing correction. Differentially expressed genes were clustered based on expression using K-means clustering (k = 4) with the Pearsons correlation distance metric within MeV (4.8.1). Results: We showed that N deprivation-induced quiescence is accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis leading to accumulation of triacylglycerol (TAG). Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of TAG stored in lipid droplets (LDs). Beside observing LD translocation into vacuoles we also provide evidence for direct interaction between the lipid droplet surface protein (NoLDSP) and autophagy-related 8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. Conclusions: This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for the development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.
2019-10-01 | GSE129245 | GEO
Project description:Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high value lipid products. First success in applying reverse genetics makes Nannochloropsis species attractive models to investigate the cell and molecular biology and biochemistry of this fascinating organism group. (Principle findings) Here we present the assembly of the 28.7 Mb genome of Nannochloropsis oceanica CCMP1779. RNA sequencing data from N-replete and N-depleted growth conditions support a total of 11,973 genes, which in addition to automatic annotation were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors and 109 transcriptional regulators were annotated. In addition, we provide protocols for the transformation of the sequenced strain. (Significance) The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols provides a blueprint for future detailed gene functional analysis and phylogenetic comparison of Nannochloropsis species by a growing academic community focused on this genus.
2012-04-04 | GSE36959 | GEO
Project description:Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3g26790, FUS3), are associated with regulation of embryo 3maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50 fold over controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased 3-4 fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation.
2016-04-19 | GSE80360 | GEO
Project description:Chromochloris zofingiensis has been considered as potential feedstock for biodiesel production with advantages such as high biomass productivity and great oil content. TAG (triacylglycerol) and astaxanthin accumulate in a well-coordinated manner in response to different stresses in C. zofingiensis. The integrated production of lipids with co-products emerges as a new research direction and is proposed to be a promising approach toward offsetting the algal biodiesel production cost. Therefore, it is suggested C. zofingiensis can serve as research models for the integrated production. Salinity stress simultaneously induced TAG and astaxanthin accumulation in C. zofingiensis. To understand the mechanism underlying TAG and astaxanthin accumulation induced by salinity stress, we applied high-throughput mRNA-sequencing in C. zofingiensis.
2020-04-27 | GSE125419 | GEO
Project description:Robust protocols and automation now enable large-scale single-cell RNA and ATAC sequencing experiments and their application on biobank and clinical cohorts. However, technical biases introduced during sample acquisition can hinder solid, reproducible results and a systematic benchmarking is required before entering large-scale data production. Here, we report the existence and extent of gene expression and chromatin accessibility artifacts introduced during sampling and identify experimental and computational solutions for their prevention.
2020-04-27 | GSE132065 | GEO