Briel Simon Carl-Philipp, Feuser Nicolas, Moldenhauer Eva Johanna, Kabisch Johannes, Neubauer Peter, Junne Stefan
Technische Universität Berlin, Department of Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany.
Technische Universität Darmstadt, Department of Biology, Computer-Aided Synthetic Biology, Darmstadt, Germany.
J Biotechnol. 2025 Jan;397:32-43. doi: 10.1016/j.jbiotec.2024.11.011. Epub 2024 Nov 17.
Digital holographic microscopy (DHM) is a label-free analytical technique for the determination of the cells' volume and their cytosolic refractive index. Here, we demonstrate the suitability of DHM for the quantification of total lipid accumulation in the oleaginous yeast Yarrowia lipolytica. Presently, microbial lipids are gaining increasing attention due to their nutritional value in feed and food applications. Their microbiological synthesis in algae and yeast is subject to optimization studies, which necessitates rapid quantification of total lipids for faster progress and the possibility of process control. So far, quantification of the total intracellular long-chain fatty acid concentration in yeast cells is time-consuming though when common chromatography for a volumetric analysis or staining and flow cytometry for a single-cell based analysis are used. This study, however, demonstrates that 3D-DHM facilitates a quasi-real-time measurement that allows for a rapid quantification of total intracellular lipid accumulation on a single-cell level without cell staining. Data from wild-type and lipid overproducing Y. lipolytica strains with specific yields of long-chain fatty acids in a range between 70 and 360 mg/gCDW show a good correlation with the optical volume determined by DHM, as the total lipid accumulation in the cell is typically well correlated with the long-chain fatty acid concentration. The results further correlate with data obtained from gas chromatography and flow cytometry of Nile Red-stained cells, which proves the reliability of DHM for lipid quantification in Y. lipolytica.
数字全息显微镜（DHM）是一种用于测定细胞体积及其胞质折射率的无标记分析技术。在此，我们证明了DHM适用于定量分析产油酵母解脂耶氏酵母中的总脂质积累。目前，微生物脂质因其在饲料和食品应用中的营养价值而受到越来越多的关注。它们在藻类和酵母中的微生物合成正在进行优化研究，这就需要快速定量总脂质，以便更快地取得进展并实现过程控制的可能性。到目前为止，使用常规色谱法进行体积分析或染色和流式细胞术进行单细胞分析时，定量酵母细胞内总细胞内长链脂肪酸浓度很耗时。然而，这项研究表明，3D-DHM有助于进行准实时测量，能够在不进行细胞染色的情况下在单细胞水平上快速定量总细胞内脂质积累。来自野生型和解脂耶氏酵母脂质高产菌株的数据，其长链脂肪酸的特定产量在70至360 mg/gCDW之间，与DHM测定的光学体积显示出良好的相关性，因为细胞中的总脂质积累通常与长链脂肪酸浓度密切相关。结果进一步与尼罗红染色细胞的气相色谱法和流式细胞术获得的数据相关，这证明了DHM用于解脂耶氏酵母脂质定量的可靠性。
