Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Audrey Bloem; Stephanie Rollero; Pauline Seguinot; Lucie Crépin; Marc Perez; Christian Picou; Carole Camarasa

doi:10.3791/56393

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

9.2K views

⸱

DOI:

10.3791/56393

⸱

January 22nd, 2018

Audrey Bloem¹ , Stephanie Rollero² , Pauline Seguinot¹ , Lucie Crépin³ , Marc Perez¹ , Christian Picou¹ , Carole Camarasa¹

¹UMR SPO, INRA, SupAgroM, Université de Montpellier, ²IWBT Stellenbosch University, ³PILI, Toulouse White Biotechnology

Summary

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Abstract

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.

We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected ¹³C- or ¹⁵N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples–facilitated by a robot-assisted system for online monitoring of fermentations–and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

Introduction

Understanding how microbial metabolism operates is a key issue for the design of efficient strategies to improve fermentation processes and modulate the production of fermentative compounds. Advances in genomics and functional genomics in these last two decades largely contributed to extending knowledge of the topology of metabolic networks in many microorganisms. Access to this information led to the development of approaches that aim for a comprehensive overview of cellular function¹. These methodologies often rely on a model-based interpretation of measurable parameters. These experimental data include, on one hand, metabolite uptake and pro....