Metabolomics is an essential discipline in industrial biotechnology. Sample preparation approaches dramatically influence data quality and, ultimately, interpretation and conclusions from metabolomic experiments. However, standardized protocols for highly reproducible metabolic datasets are limited, especially for the fungal cell factory Aspergillus niger. Here, an improved liquid chromatography‐tandem mass spectrometry‐based pipeline for A. niger metabolomics is developed. It is found that fast filtration with liquid nitrogen is more suitable for cell quenching, causing minimal disruption to cell integrity, and improved intracellular metabolite recovery when compared to cold methanol quenching approaches. Seven solutions are evaluated for intracellular metabolite extraction, and found acetonitrile/water (1:1, v/v) at −20 °C, combined with boiling ethanol extraction protocols, showed unbiased metabolite profiling. This improved methodology is applied to unveil the dynamic metabolite profile of one citrate over‐producing A. niger isolate under citrate fermentation. Citrate precursors, especially pyruvate, oxaloacetate, and malate, are maintained at a relatively high intracellular level, which can be necessary for high citrate synthesis flux. Glutamine shows a similar trend compared to citrate production, suggesting glutamine may be involved in intracellular pH homeostasis. Taken together, this study delivers a highly standardized and improved metabolomics methodology and paves the way for systems metabolic engineering in biotechnologically important fungi.

中文翻译：

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样品制备方法的全面改进有助于黑曲霉的动态代谢组学

代谢组学是工业生物技术中必不可少的学科。样品制备方法极大地影响数据质量，最终影响代谢组学实验的解释和结论。然而，用于高度可再现的代谢数据集的标准化方案是有限的，尤其是对于真菌细胞工厂黑曲霉而言。这是一条改进的基于液相色谱串联质谱的黑曲霉管道代谢组学的发展。发现与冷甲醇淬灭方法相比，用液氮快速过滤更适合于细胞淬灭，对细胞完整性的破坏最小，并改善了细胞内代谢物的回收率。对七种溶液进行了细胞内代谢物提取评估，发现在-20°C的乙腈/水（1：1，v / v）与沸腾的乙醇提取方案结合后，代谢物谱图无偏。这种改进的方法学被用于揭示一种柠檬酸盐生产过量的黑曲霉的动态代谢产物概况在柠檬酸发酵下分离。柠檬酸盐前体，特别是丙酮酸，草酰乙酸和苹果酸，被维持在较高的细胞内水平，这对于高柠檬酸盐合成通量可能是必要的。与柠檬酸生产相比，谷氨酰胺显示出相似的趋势，表明谷氨酰胺可能参与细胞内pH稳态。总之，这项研究提供了高度标准化和改进的代谢组学方法，为生物技术重要的真菌中的系统代谢工程铺平了道路。