In vivo online monitoring of intracellular lipid accumulation in Ustilago maydis

Single cell oils produced in microorganisms constitute appealing alternatives to plant oils. Oleaginous fungi accumulate triacylglycerols in lipid droplets (LD). Their biosynthesis is typically induced under nitrogen limitation. We exploit the fungal model Ustilago maydis for oil production. The stain BODIPY can be used to track LD formation during cultivation but this expensive compound is only affordable at small-scale. Therefore, mutant screening for optimization of oil production and composition would benefit from an inexpensive online monitoring system. Accordingly, we aimed at developing an intrinsic reporter that is suitable to track oil formation even in larger cultures. From three tested candidates, the potential delta(24)-sterol C-methyltransferase Erg6 turned out as the best reporter. Fluorescence microscopy confirmed its localization at the LD membrane. After optimization, Erg6 fused to mKate2 expressed from a promoter derived from glycolipid biosynthesis showed a good correlation of fluorescence with oil accumulation. Time course experiments in micro-cultivators demonstrated that the fluorescence read-out can be used to track oil formation starting at the onset of nitrogen limitation to approximate the LD amount. In essence, our study introduces a biosensor for oil monitoring, that can easily be transferred to other oleaginous yeasts.hosted on: https://git.nfdi4plants.org/projects/3204

微生物来源的单细胞油脂是极具吸引力的植物油脂替代方案。产油真菌可在脂滴（lipid droplets, LD）中积累三酰甘油，其生物合成通常在氮限制条件下被诱导。本研究以真菌模式菌株玉米黑粉菌（Ustilago maydis）作为油脂生产宿主。染色剂BODIPY可用于追踪培养过程中的脂滴形成，但该化合物成本高昂，仅能在小规模实验中使用。因此，若要开展优化油脂产量与组分的突变体筛选，亟需一款低成本的在线监测系统。基于此，本研究旨在开发一种内源报告基因，即便在大规模培养体系中也能有效追踪油脂形成过程。在三个候选蛋白中，delta(24)-甾醇C-甲基转移酶（delta(24)-sterol C-methyltransferase, Erg6）被证实为最优报告基因。荧光显微镜成像证实其定位于脂滴膜。经优化后，由糖脂生物合成相关启动子驱动表达的mKate2融合Erg6蛋白，其荧光信号与油脂积累量呈现良好的相关性。微型培养罐中的时间进程实验表明，该荧光读数可用于追踪氮限制起始阶段的油脂形成过程，从而估算脂滴数量。综上，本研究开发了一款油脂监测生物传感器，该传感器可便捷地应用于其他产油酵母。数据集托管于：https://git.nfdi4plants.org/projects/3204