Comprehensive Improvement of Sample Preparation Methodologies Facilitates Dynamic Metabolomics of Aspergillus niger
Comprehensive Improvement of Sample Preparation Methodologies Facilitates Dynamic Metabolomics of Aspergillus niger
复制标题
样品制备方法的全面改进促进黑曲霉动态代谢组学
DOI：
10.1002/biot.201800315
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发表时间：
2019
影响因子：
4.7
通讯作者：
Ma Yanhe
中科院分区：
文献类型：
--
作者：
Zheng Xiaomei;Yu Ji;ong;Cairns Timothy C;Zhang Lihui;Zhang Zhidan;Zhang Qiongqiong;Zheng Ping;Sun Jibin;Ma Yanhe
代谢组学是工业生物技术的一门重要学科。样品制备方法显著影响数据质量，并最终影响代谢组学实验的解释和结论。然而，高度可重复的代谢数据集的标准化协议是有限的，特别是对于真菌细胞尼日尔。在这里，一个改进的液相色谱串联质谱为基础的管道A。开发了尼日利亚代谢组学。已发现，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，对细胞完整性造成最小的破坏，并且改善了细胞内代谢物的回收。评价了7种溶液的细胞内代谢物提取，发现在−20 °C下的乙腈/水（1：1，v/v），结合沸腾乙醇提取方案，显示出无偏倚的代谢物谱。这种改进的方法用于揭示一种柠檬酸盐过度生产A的动态代谢产物谱。nigerisolate在柠檬酸发酵。柠檬酸盐前体，特别是丙酮酸盐、草酰乙酸盐和苹果酸盐，保持在相对高的细胞内水平，这对于高柠檬酸盐合成通量是必需的。谷氨酰胺显示出与柠檬酸盐生产类似的趋势，表明谷氨酰胺可能参与细胞内pH稳态。总之，这项研究提供了一个高度标准化和改进的代谢组学方法，并为生物技术重要真菌的系统代谢工程铺平了道路。
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 factoryAspergillus niger. Here, an improved liquid chromatography‐tandem mass spectrometry‐based pipeline forA. nigermetabolomics 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‐producingA. nigerisolate 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.