High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica
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ABSTRACT:
PROVIDER: PRJNA702802 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13739509_1.fastq.gz | Fastqsanger.gz | |||
| SRR13739509_2.fastq.gz | Fastqsanger.gz | |||
| SRR13739510_1.fastq.gz | Fastqsanger.gz | |||
| SRR13739510_2.fastq.gz | Fastqsanger.gz | |||
| SRR13739511_1.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Nannochloropsis oceanica CCMP1779 is a marine unicellular stramenopile and an emerging reference species for basic research on oleogenic microalgae with biotechnological relevance. We investigated its physiology and transcriptome under light/dark cycles. We observed oscillations in lipid content and a predominance of cell division in the first half of the dark phase. Globally, more than 60% of the genes cycled in N. oceanica CCMP1779, with gene expression peaking at different times of the day. Interestingly, the phase of expression of genes involved in certain biological processes was conserved across photosynthetic lineages. Furthermore, in agreement with our physiological studies we found the processes of lipid metabolism and cell division enriched in cycling genes. For example, there was tight coordination of genes involved in the lower part of glycolysis, fatty acid synthesis and lipid production at dawn preceding lipid accumulation during the day. Our results suggest that diel lipid storage plays a key role for N. oceanica CCMP1779 growth under natural conditions making this alga a promising model to gain a basic mechanistic understanding of triacylglycerol production in photosynthetic cells. Our data will help the formulation of new hypotheses on the regulation transcriptional control of cell growth and metabolism in Nannochloropsis. Nannochloropsis oceanica CCMP was entrained to 12:12 light:dark cycles and biological replicates collected every 3 hours for a cycle for a total of 16 samples.
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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
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Project description:Transcriptional regulation in response to nitrogen stress for one ant two days, and epigenome-association with phenotypic plasticity of lipid metabolism are uncovered in marine microalga Nannochloropsis oceanica IMET1.
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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.
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