Comprehensive Improvement of Sample Preparation Methodologies Facilitates Dynamic Metabolomics ofAspergillus niger

Abstract

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.

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

“…In order to elucidate the impact of pyrG disruption on the A. niger intracellular metabolite profile, metabolomics analyses of parent strain D353 and pyrG deficient mutant D353.8 were conducted by our established LC-MS/MS pipeline [35]. Samples were taken at the end of citric acid fermentation in shake flasks (Fig.…”

“…Citrate fermentation was carried out using the liquefied corn media [35]. The final concentration of 1 × 10 5 spores/mL was inoculated in 20 mL liquefied corn media with different concentration of Dox in 100 mL shake flasks, which were cultivated at 34 °C and 220 rpm for 96 h. The weight of the shake flasks were measured before and after the citric acid fermentation to eliminate measurement errors caused by evaporation.…”

“…The samples for intracellular metabolite profiling were prepared and detected based on the standardized and improved LC-MS/MS metabolomics methodology [35]. Briefly, 5 mL (± 0.5) of A. niger mycelial culture was fast filtered by a -20 °C pre-cooled vacuum filter with six layers Miracloth (CalBiochem, Merck, Darmstadt, Germany).…”

“…The second mixture was centrifuged again and the supernatant extraction were mixed with the first supernatant and centrifuged at 12,000 rpm for 30 min at 0 °C. The supernatant was then collected, freeze dried and detected by the LC-MS/MS platform containing Ultra-performance liquid chromatography (UPLC) 30A (Shimadzu, Kyoto, Japan) and TripleTOF ™ 6600 mass spectrometer (Applied Biosystem Sciex, USA) as described previously [35]. The LC-MS/MS data were normalized by sample weight, and then metabolites involved in central metabolism were identified according to the protocol described [35].…”

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

“…Among these “omics” approaches, the metabolome covers all metabolites in an organism, which is governed by cellular‐regulatory processes sensitive to genetic and/or environmental perturbations . Comparative metabolomics approaches can provide insights into cellular metabolism by providing a comprehensive analysis of all metabolites in a biological system and can reveal metabolic relationships and metabolite responses . In addition, complementary to metabolite concentrations, it also provides an opportunity for linking metabolites and their pathways .…”