Exploring the Glucose Fluxotype of the E. coli y-ome Using High-Resolution Fluxomics

Abstract

We have developed a robust workflow to measure high-resolution fluxotypes (metabolic flux phenotypes) for large strain libraries under fully controlled growth conditions. This was achieved by optimizing and automating the whole high-throughput fluxomics process and integrating all relevant software tools. This workflow allowed us to obtain highly detailed maps of carbon fluxes in the central carbon metabolism in a fully automated manner. It was applied to investigate the glucose fluxotypes of 180 Escherichia coli strains deleted for y-genes. Since the products of these y-genes potentially play a role in a variety of metabolic processes, the experiments were designed to be agnostic as to their potential metabolic impact. The obtained data highlight the robustness of E. coli’s central metabolism to y-gene deletion. For two y-genes, deletion resulted in significant changes in carbon and energy fluxes, demonstrating the involvement of the corresponding y-gene products in metabolic function or regulation. This work also introduces novel metrics to measure the actual scope and quality of high-throughput fluxomics investigations.

Fluxomics - New Metabolomics Approaches to Monitor Metabolic Pathways

“…For instance, secondary metabolites (usually existing at very low concentrations) are beyond the detection limit of NMR spectroscopy, while for volatile molecules can be detected by GC-MS combined with the mass spectrum and retention time ( Emwas et al, 2015 ; Kohlstedt and Wittmann, 2019 ). Thus, integrating NMR spectroscopy with MS methods is important to give more comprehensive analysis ( Fan et al, 2014 ; Elbaz et al, 2015 ; Emwas and Bjerrum, 2015 ; Sá et al, 2017 ; Bergès et al, 2021 ).…”

“…In the recent studies, Bergès et al utilized both NMR and MS approaches to obtain high resolution fluxotypes for huge numbers of a strains in a library. They define fluxotype as “ the particular distribution of metabolic fluxes measured for a given strain under given physiological conditions ” ( Bergès et al, 2021 ). The authors studied the fluxotype of 180 different E. coli strains with deleted y-genes.…”

“…Bacteria were grown in 13 C labeled glucose as a single source of carbon while monitoring metabolic fluxes. Deletion of two y-genes led to a significant modification of metabolic fluxes indicating the role of the studied genes in metabolic regulation ( Bergès et al, 2021 ).…”

An optimization method for untargeted MS-based isotopic tracing investigations of metabolism

“…These cultures were performed in 48 15 ml bioreactors under controlled growth conditions using a robotic platform (Freedom EVO 200, Tecan), with collection of labelled samples (biomass or cultivation medium) at defined culture times or optical densities. This cell culture robot and its operation are described in detail in Heux et al ( 2014 ) and Bergès et al ( 2021 ).…”

Metabolomics and modelling approaches for systems metabolic engineering

“…The use of liquid handling technologies also allows for good reproducibility of samples extracted and is therefore vital for high throughput sample preparation ( Liu et al, 2019a ). Cultivation devices such as microtiter plates or micro-bioreactors have also been integrated to robotic cultivation platforms, enabling growth parameters for cell cultures to be fully automated and controlled ( Long et al, 2014 ; Faust et al, 2014 ; Unthan et al, 2015 ; Heux et al, 2014 ; Bergès et al, 2021 ).…”