Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Biotechnol J. 2019 Mar;14(3):e1800315. doi: 10.1002/biot.201800315. Epub 2018 Sep 20.
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.
代谢组学是工业生物技术的一个重要学科。样品制备方法会显著影响数据质量，最终影响代谢组学实验的解释和结论。然而，对于真菌细胞工厂黑曲霉来说，高度可重复的代谢数据集的标准化方案是有限的。在这里，开发了一种改进的基于液相色谱-串联质谱的黑曲霉代谢组学分析方法。研究发现，与冷甲醇淬灭方法相比，液氮快速过滤更适合细胞淬灭，对细胞完整性的破坏最小，并且可以提高细胞内代谢物的回收率。评估了七种用于细胞内代谢物提取的溶液，发现-20°C 下的乙腈/水（1:1，v/v）与沸腾乙醇提取方案相结合，可进行无偏的代谢物分析。该改进的方法学用于揭示柠檬酸高产黑曲霉分离株在柠檬酸发酵过程中的动态代谢谱。柠檬酸前体，特别是丙酮酸、草酰乙酸和苹果酸，在细胞内保持相对较高的水平，这对于高柠檬酸合成通量可能是必需的。谷氨酰胺与柠檬酸的产生呈相似趋势，表明谷氨酰胺可能参与细胞内 pH 稳态。总的来说，本研究提供了一种高度标准化和改进的代谢组学方法，为生物技术中重要真菌的系统代谢工程铺平了道路。
