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

An N T Phan,Lars M Blank

doi:10.3389/fmolb.2020.00211

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.

Highlights

Ustilago maydis is a maize pathogen that causes corn smut (Brefort et al, 2009), a plant disease reducing cereal production
U. maydis, which grows in its haploid form non-filamentous (Kubicek et al, 2011), gains attention in biotechnology due to its capability of using sustainable substrates to Ustilago maydis GC-MS-Based Metabolomics produce valuable chemicals of industrial interest such as mannitol, erythritol, mannosylerythritol lipids (MELs), ustilagic acid, itaconic acid, malic acid, and hydroxyparaconic acid (Geiser et al, 2014)
While quenching is crucial to stop cell metabolism quicker than the turnover of metabolites, washing is utilized to remove extracellular compounds from the culture medium. These steps have to compromise to keep the metabolites lost at a minimum

Summary

Introduction

Ustilago maydis is a maize pathogen that causes corn smut (Brefort et al, 2009), a plant disease reducing cereal production. Genome sequencing (Kämper et al, 2006) revealed that approximately 10% of U. maydis proteins were highly conserved in humans, which had lower similarity or did not even exist in the yeast Saccharomyces cerevisiae (Münsterkötter and Steinberg, 2007). This finding makes U. maydis a unique model organism for many fundamental studies that yeast models do not offer such as endocytosis, long-distance mRNA transport, cell signaling, microtubule organization, and polarized growth (Steinberg and Perez-Martin, 2008; Etxebeste and Espeso, 2016; Haag et al, 2019). To study the genotypephenotype relationship in U. maydis, it is crucial to develop a robust protocol for metabolomics

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Results