A novel platform for automated high-throughput fluxome profiling of metabolic variants
Introduction
In the field of biotechnology, metabolic engineering and synthetic biology, fluxomics has been identified as a key analytical technology (Ellis and Goodacre, 2012, Feng et al., 2010, Sanford et al., 2002, Stephanopoulos, 1999) not only for the rational design of cells but also for comprehensive understanding of the link between genotype and phenotype. Fluxomics, i.e. the cell-wide quantification of metabolic fluxes, reveals the actual operation of metabolic networks in given environmental conditions, resulting from the integrated flow of interactions between all molecular components - genes, mRNAs, proteins and metabolites (Sauer, 2004, 2006). Hence, metabolic flux analysis is the most comprehensive description of the metabolic phenotype at the cellular level (Wittmann and Portais, 2013). In practice, 13C-fluxomics is still a tedious and rather time consuming process. The method combines both 13C-labelling experiments and mathematical modelling of biochemical networks. Cells are gown on 13C-labelled substrates to metabolic and isotopic steady state and the labelling patterns of metabolites are monitored by mass spectrometry (MS), nuclear magnetic resonance (NMR), or both. The labelling information can be collected on metabolic end-products such as protein-bound amino acids, which accumulate to larger extents than true metabolic intermediates (Sauer, 2004, Szyperski, 1995, Wiechert, 2001). Combined with quantitative physiological data and a detailed metabolic model of metabolism, the labelling patterns give access to the in vivo reaction rates (i.e. fluxes) associated with the cellular network of an organism. When this information is not available, multivariate statistics can be applied to the isotopic data to provide detailed phenotyping of biological systems without any prior knowledge (Raghevendran et al., 2004, Zamboni and Sauer, 2004).
The rapid expansion of fluxomics and its areas of application is driving the need for high-throughput approaches to enable comprehensive metabolic investigations of a growing number of organisms, engineered mutants and physiological conditions (Ellis and Goodacre, 2012). Significant advances have been made in the field in the last decade. Miniaturized cell cultivation systems have been used to perform 13C-labelling experiments with less effort and cost in labelled substrate (Balcarcel and Clark, 2003, Betts and Baganz, 2006, Ge et al., 2006, Girard et al., 2001, Huber et al., 2009, Isett et al., 2007, Kocincova et al., 2008, Tang et al., 2009). The sensitivity, speed, and robustness of both NMR-based and MS-based isotopic analysis have been improved (Boisseau et al., 2013, Cahoreau et al., 2012, Fan and Lane, 2008, Fischer et al., 2004, Giraudeau et al., 2012, Giraudeau et al., 2011, Massou et al., 2007a, Peng, 2012). Tools have been developed for large scale isotopic data processing (Millard et al., 2012, Poskar et al., 2012) as along with improved algorithms and software for flux calculation and statistical analysis of isotopic data (Antoniewicz et al., 2007, Quek et al., 2009, Raghevendran et al., 2004, Sokol et al., 2012, Weitzel et al., 2012, Zamboni et al., 2005, Zamboni and Sauer, 2004). These developments have allowed large-scale flux analysis to be applied to microorganisms (Amador-Noguez et al., 2010, Blank et al., 2005, Fischer and Sauer, 2005, Fischer et al., 2004, Haverkorn van Rijsewijk et al., 2011, Wittmann et al., 2004) and mammalian cells (Munger et al., 2008).
However, despite all the above improvements, HT fluxomics is still in early development. In particular, there is no existing HT fluxomics platform which combines all experimental and in silico steps of the complete workflow in a single and fully integrated manner. The design and development of such a platform is hindered by several technical challenges that need to be addressed in parallel. This includes (1) the tight control of cultivation parameters to ensure metabolic and isotopic steady state; (2) the automated monitoring of growth parameters to collect labelled material once steady state is achieved (3) parallel cultivation to increase throughput; (4) miniaturization of working volumes to reduce the cost of labelled substrates and to facilitate the parallel processes; (5) automated, rapid, parallel sampling of labeled material to avoid degradation of the metabolites; (6) rapid, sensitive measurement of isotopic profiles to cope with both the small amounts of biological material and the large number of samples; (7) automated extraction of labeling information from raw analytical data, which today is still mainly done manually, thereby saving time and effort and improving data robustness (8), data interpretation tools that provide valuable metabolic information in a high throughput manner, i.e. with reduced user supervision.
For the first time, these challenges were met with the development of a fully integrated solution for fluxome analysis that combines a robotic cultivation and sampling workstation for 13C-labelling experiments with NMR-based isotopic profiling, and tools for processing and interpreting isotopic data. The automation, parallelization, optimization and integration of all steps in the workflow are described in detail. As a proof of concept, the new platform was applied to a set of Escherichia coli mutants with varying levels of a single enzyme and grown on two different 13C-labelled carbon sources. The power of the overall approach to provide discriminating metabolic information for a large number of mutants and conditions as well as its value in generating valuable metabolic knowledge was demonstrated.
Section snippets
Materials and methods
Fig. 1 is a schematic diagram of the complete workflow developed in this study.
Development of a robotic cultivation and sampling system
A workstation enabling (i) the automated parallel cultivation of bacteria with 13C-labelled substrates and (ii) the automated sampling of labelled metabolites under steady state conditions was designed and constructed. Cell cultivation was automated, parallelized and miniaturized by incorporating a block of 48 micro-scale bioreactors (Kusterer et al., 2008, Puskeiler et al., 2005) in the automatic workstation. This system allows fully automated and reproducible monitoring of growth, pH and
Conclusion
The integrated fluxome platform presented here is a first but critical step towards robotic screening of quantitative metabolic phenotypes. Due to the parallelization and automation of the workflow, significant improvements in throughput, robustness and release of resources were achieved. The 80 flux data collected on E. coli G6PDH-modulation mutants were generated in four days, excluding preparation time but including all automated and manual steps from cultivation to calculated fluxes. This
Acknowledgments
MetaToul (Metabolomics & Fluxomics Facitilies, Toulouse, France, www.metatoul.fr) and its staff members Edern Cahoreau and Lindsay Peyriga are gratefully acknowledged for technical support and access to NMR. MetaToul is part of the national infrastructure MetaboHUB (The French National infrastructure for metabolomics and fluxomics, www.metabohub.fr, MetaboHUB-ANR-11-INBS-0010). MetaToul is supported by the Région Midi-Pyrénées, the European Regional Development Fund, the SICOVAL, the
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