GC-MS-Based Metabolomics for the Smut Fungus Ustilago maydis: A Comprehensive Method Optimization to Quantify Intracellular Metabolites

Abstract

Ustilago maydis, a smut fungus, is an appealing model in fundamental research and an upcoming cell factory for industrial biotechnology. The genome of U. maydis has been sequenced and some synthesis pathways were biochemically described; however, the operation of the cellular metabolic network is not well-characterized. Thus, we conducted a comprehensive study to optimize the sample preparation procedure for metabolomics of U. maydis using GC-MS/MS. Due to the unique characteristics of U. maydis cell culture, two quenching solutions, different washing steps, eight extraction methods, and three derivatization conditions have been examined. The optimal method was then applied for stable isotope-assisted quantification of low molecular weight hydrophilic metabolites while U. maydis utilized different carbon sources including sucrose, glucose, and fructose. This study is the first report on a methodology for absolute quantification of intracellular metabolites in U. maydis central carbon metabolism such as sugars, sugar phosphates, organic acids, amino acids, and nucleotides. For biotechnological use, this method is crucial to exploit the full production potential of this fungus and can also be used to study other fungi of the family Ustilaginaceae.

Ustilaginaceae Biocatalyst for Co-Metabolism of CO2-Derived Substrates toward Carbon-Neutral Itaconate Production

“…Furthermore, deletion of fuz7 enables a stable yeast-like growth [ 17 ]. Moreover, a metabolomics method focusing on the central carbon metabolism has recently been developed for U. maydis , which can be applied to investigate the cellular metabolic network and support metabolic engineering strategy [ 62 ].…”

Metabolomics and modelling approaches for systems metabolic engineering

“…However, this additional derivatization step can result in analyte loss and/or artifact formation ( Kanani et al, 2008 ). Central carbon metabolites and amino acids are easily analyzed using GC-MS. Organic acids, sugar phosphates and alcohols, are usually derivatized in pyridine methoxyamine solution followed by trimethylsilylation ( Phan and Blank, 2020 ). However, metabolites that are non-volatile or thermally unstable cannot be analyzed by the GC and requires LC instead.…”

GC‐MS‐based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect

“…[18,19] Many of these studies employ derivatization strategies to investigate polar metabolites with low molecular weight (e. g., sugars, amino acids, and organic acids) that are mainly associated with central biosynthetic pathways. [20][21][22][23][24] Yet, employing solvents that extract a wide spectrum of organic compounds (e. g., dichloromethane) [25] may expand the use of GC-MS analyses into a broad-ranging approach for a variety of medium-polar and non-polar compounds (e. g., sterols, triglycerides, phospholipids, and fatty acids).…”