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

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. Transcriptome of an insertional loss-of-function mutant for gene NO06G03670 (strain HLM23) compared to the wild type, cultivated under favorable growth conditions.

产油微藻（oleaginous microalgae）被认为是可持续生产高价值脂质与生物燃料原料的极具潜力的平台。然而当前的脂质产率过低，无法实现经济上可行的规模化生产。通过基因工程改造微藻菌株，可提升脂质产率。针对工业应用相关的拟微球藻属（Nannochloropsis）的菌株改造策略收效甚微，因为该属的绝大多数基因缺乏功能注释，阻碍了我们对脂质代谢及其调控机制的认知。为获取基础认知并为脂质代谢的基因工程改造提供靶点，本研究旨在挖掘海洋拟微球藻（Nannochloropsis oceanica）中与中性脂质（NL）含量提升相关的新型基因。因此，我们构建了海洋拟微球藻的随机基因敲除（gene knockout, KO）插入诱变文库，并通过五轮荧光激活细胞分选（fluorescence-activated cell sorting）筛选得到高脂质突变株（HLM）菌株。在适宜生长条件下，多株突变株的中性脂质含量较野生型菌株有所提升。借助包含IIS型限制性内切酶MmeI的改良盒式PCR策略，我们确定了五株最具潜力突变株的目标基因敲除位点。其中一株极具潜力的突变株HLM23的NO06G03670基因发生插入断裂，该基因编码一个假定的APETALA2类转录因子（APETALA2-like transcription factor）。HLM23的生长速率未受影响，光合性能有所提升，其中性脂质含量可达干细胞重的30%，较野生型提升40%。转录组测序结果显示，该突变株中与质体脂肪酸生物合成、糖酵解以及卡尔文-本森-巴斯汉循环（Calvin–Benson–Bassham cycle）相关的基因均出现转录上调。本数据集为适宜生长条件下，基因NO06G03670插入失活突变株（HLM23）与野生型菌株的转录组对比数据。