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High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica

ABSTRACT: 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.

ORGANISM(S): Nannochloropsis oceanica strain IMET1

PROVIDER: GSE167058 | GEO | 2021/05/13

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.

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High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica

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