Background

Microalgae are considered a promising platform for sustainable lipid production. Despite this, productivities need to be improved to facilitate an economically viable production process. This can be achieved through strain improvement, for instance by genetic engineering. Strain improvement strategies often deploy high-throughput screening platforms i.a. involving single-cell methodologies such as fluorescence-activated cell sorting (FACS) for the identification and isolation of better-performing strains. The heterokont microalga Nannochloropsis is a prospective candidate for the industrial production of lipids. Previous studies have reported the isolation of high lipid-producing Nannochloropsis strains by combining qualitative staining of lipid bodies using the fluorophoric dye BODIPY with FACS methodology. However, it has never been investigated how cellular physiology and different staining conditions hamper the reproducibility of this method as a quantitative screening procedure.

Results

Here we report the development of an optimized single cell lipid screening procedure for Nannochloropsis oceanica. Systematic assessment of different staining conditions revealed that treatment with 6% DMSO and 1.2 μg ml⁻¹ BODIPY for 15 min is ideal for staining neutral lipids in an exponentially growing culture of N. oceanica. Cultures that are overproducing lipids, for example after exposure to external stimuli such as nutrient deprivation stress, require treatment with 10% DMSO and 1.2–1.6 μg ml⁻¹ BODIPY for 36 min to facilitate complete staining of lipid bodies. We verify that DMSO is required to permeabilize the particularly tough cell barrier of Nannochloropsis and we show that exposure to 10% DMSO does not affect cell viability. Increasing concentrations of BODIPY, however, correlated with a decrease in viability when screening stressed cultures. Using the optimized protocol, reproductive viabilities can be expected to be ~91% and 83–82% for non-stressed and stressed cultures respectively. The optimized procedure allows for a quantitative prediction of cellular neutral lipids (R²= 0.981), as determined by comparison to results obtained through a reference procedure.

中文翻译：

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使用BODIPY 505/515优化高通量微藻海洋小球藻的脂质筛选

背景

微藻被认为是可持续脂质生产的有前途的平台。尽管如此，仍需要提高生产率以促进经济上可行的生产过程。这可以通过菌株改良，例如通过基因工程来实现。菌株改良策略通常会部署高通量筛选平台，例如使用单细胞方法，例如荧光激活细胞分选（FACS）来鉴定和分离性能更好的菌株。杂种微藻Nannochloropsis是脂质工业生产的潜在候选对象。先前的研究报道了高产脂质的拟南芥的分离通过使用荧光染料BODIPY和FACS方法对脂质体进行定性染色，可以对菌株进行鉴定。然而，从未研究过细胞生理学和不同的染色条件如何阻碍这种方法作为定量筛选程序的可重复性。

结果

在这里我们报告了Nannochloropsis oceanica优化的单细胞脂质筛选程序的发展。对不同染色条件的系统评估显示，用6％DMSO和1.2μgml ^-1 BODIPY处理15分钟，对于在指数增长的海生猪笼草中中性脂质染色是理想的。例如，在暴露于外部刺激（如营养剥夺压力）后，产生过量脂质的培养物需要用10％DMSO和1.2–1.6μgml ^-1 BODIPY处理36分钟，以促进脂质体的完全染色。我们验证了需要DMSO来透化Nannochloropsis的特别坚硬的细胞屏障并且我们表明暴露于10％DMSO不会影响细胞活力。然而，当筛选应激培养物时，BODIPY浓度的增加与活力的降低相关。使用优化的实验方案，对于非应激培养和应激培养，其繁殖力预计分别约为91％和83-82％。优化的程序可以定量预测细胞中性脂质（R ² = 0.981），与通过参考程序获得的结果相比较即可确定。