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NHEJ-mediated insertional mutagenesis reveals a novel target for enhancing fatty alcohols production in Yarrowia lipolytica

ABSTRACT: NHEJ-mediated insertional mutagenesis reveals a novel target for enhancing fatty alcohols production in Yarrowia lipolytica

PROVIDER: PRJNA821898 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18573393_1.fastq.gz	Fastqsanger.gz
	SRR18573393_2.fastq.gz	Fastqsanger.gz
	SRR18573394_1.fastq.gz	Fastqsanger.gz
	SRR18573394_2.fastq.gz	Fastqsanger.gz
	SRR18573395_1.fastq.gz	Fastqsanger.gz

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NHEJ-mediated insertional mutagenesis reveals a novel target for enhancing fatty alcohols production in Yarrowia lipolytica

Project description:We employed omics analyses to identify the new target discovered, enabling us to systematically reveal changes in gene transcript levels resulting from the disruption of new targets that can be engineered to enhance fatty alcohols production in Yarrowia lipolytica. Transcriptomic results indicated that disruption of the new target A1 resulted in significantly increased transcript levels of genes involved in protein synthesis processes, which was strongly enhanced.

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

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

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:Total RNA versus genomic DNA hybridization on custom arrays designed for all Yarrowia lipolytica genes Total RNA was collected in mid-log phase from Yarrowia lipolytica cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Yarrowia lipolytica.

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