GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments

How metabolomics is used to support the MetaPath solution through metabolic profiling of cheese fermentation industry ecosystem models ?

International audienceConsumer expectations and societal evolutions are at the heart of the concerns of fermentation professionals. The organoleptic properties of their products are closely correlated to the manufacturing process but also to its composition specifically the microbial ecosystems introduced to produce this fermentation. In this context, the Bpifrance MetaPath project aims to reconstruct the metabolic maps of these microbial ecosystems in order to guide the industrialist in the selection of microbial strains at the time of product design. As a partner, the MetaToul platform aims to perform metabolic profiling of ecosystems in the cheese model of industrial partner Bel. The sample preparation and analysis methods developed on the MetaToul platform on this complex matrix allow a complete and precise study of the central and energetic metabolism (~80 metabolites) of these ecosystem models. It was validated with the addition of IDMS on repeatability parameters, extraction yield and matrix effect. The samples produced for metabolomics are analyzed on the following analytical systems: ion chromatography coupled to an LTQ-Orbitrap-Velos (Thermo) for the analysis of central energetic metabolism, liquid chromatography coupled to a QExactive+ (Thermo) for the analysis of amino acids, coenzymes A and the mevalonate pathway. The metabolic profiling data produced on the different ecosystem models of the partner will be integrated with others omics data into the MetaPath solution by Abolis/ Microbiome Studio partner in order to reconstruct active metabolic maps, true identity cards of the metabolic activity of microbial ecosystems in the fermentation of cheese matrix

Miniaturization of a metabolomics and fluxomics sample preparation workflow

International audienceOne of the current challenges in metabolomics and fluxomics is to gain access to the cellular heterogeneity to have access to subpopulations of cells or even of a single cell metabolism. In its second component, the national metabolomics and fluxomics infrastructure MetaboHUB is developing a work package with the objective of being able to perform single cell experiments in metabolomics and fluxomics. Unlike other omics like transcriptomics, Single-cell metabolomics is still in its infancy and only very few attempts of single cell fluxomics have been reported so far. In this poster, focus will be done on the developments realized for metabolomics and fluxomic sample preparation step. Indeed, methods conventionally used involve working with several hundred microliters or even several milliliters. An intermediate step of this workpackage is to be able to work on small volumes of samples in order to analyze matrices for which we have few amounts available or to concentrate classical samples and have access to minority metabolites. For this purpose, we adapted our current robotic systems in order to allow precision work with smaller volumes. The developments made allow us today to aspirate and dispense 2 µL with a CV less than 10%. In a second hand, developments of new robotic system protocols were done in order to prepare miniaturized NMR samples. With the use of new capillary needles, we can automatically filled NMR tubes of 1.7 and 1mm inner diameters. This opens up new perspectives, notably by reducing the volume of samples required for NMR analysis

Metabolic profiles of complex fermentation matrices from cream and bakery industry

International audienceIntroduction Microbial ecosystems are key factors in food production by fermentation. The metabolic potential of ecosystems is very important and varies depending on the ecosystem composition and the metabolic capacity of the different partners. The construction of the metabolic map of an ecosystem is an important tool to understand, develop and use these metabolic capacities. In this context the MetaPath Bpi France project aims to develop an integrated solution allowing this modeling. In this context, the MetaToul platform aims to develop methods adapted to different food matrices for study of the metabolism, the most accurate and global possible of these different cellular ecosystems. Our main action is first of all to develop, on these complex fermented cream and sourdoughs, the extraction and global analysis of the central and energetic metabolism

Reaching metabolomic niches through miniaturization of 1 H-NMR experiments.

International audienceNMR spectroscopy is a great source of information for metabolomics studies. However, one major drawback of NMR is its lack of sensitivity which limits its use for small samples. In such cases, the samples have to be diluted and the experiment time lengthened to accumulate enough signal, which is not optimal. Miniaturization of NMR experiments in the framework of the REACT-EU OCSSIGEN project could be the way to overcome these limitations and reach new metabolomic applications

How metabolomics is used to support the MetaPath solution through metabolic profiling of sourdough fermentation industry ecosystem models?

Consumer expectations and societal evolutions are at the heart of the concerns of fermentation professionals. The organoleptic properties of their products are closely correlated to its manufacturing process but also to its composition specifically the microbial ecosystems introduced to produce this fermentation. In this context, the Bpifrance MetaPath project aims to reconstruct the metabolic maps of these microbial ecosystems in order to guide the industrialist in the selection of microbial strains at the time of product design. As a partner, the MetaToul platform aims to perform metabolic profiling of ecosystems in the sourdough model of industrial partner Lesaffre. The sample preparation and analysis methods developed on the MetaToul platform on this complex matrix allow a complete and precise study of the central and energetic metabolism (~80 metabolites) of these ecosystem models. It was validated with the addition of IDMS on repeatability parameters, extraction yield and matrix effect. The samples produced for metabolomics are analyzed on the following analytical systems: ion chromatography coupled to an LTQ-Orbitrap-Velos (Thermo) for the analysis of central energetic metabolism, liquid chromatography coupled to a QExactive+ (Thermo) for the analysis of amino acids, coenzymes A and the mevalonate pathway. The metabolic profiling data produced on the different ecosystem models of the partner will be integrated with others omics data into the MetaPath solution by Abolis/ Microbiome Studio partner in order to reconstruct active metabolic maps, true identity cards of the metabolic activity of microbial ecosystems in the fermentation of sourdough matrix

Document for adding numbered structures into PeakForest

This document is a standard operating procedure for the PeakForest infrastructure (peakforest.org)Its contains instructions for numbering molecules in relation with Nuclear magnetic resonance data

Metabolic flux analysis in Ashbya gossypii using 13C-labeled yeast extract: industrial riboflavin production under complex nutrient conditions

Abstract Background The fungus Ashbya gossypii is an important industrial producer of the vitamin riboflavin. Using this microbe, riboflavin is manufactured in a two-stage process based on a rich medium with vegetable oil, yeast extract and different precursors: an initial growth and a subsequent riboflavin production phase. So far, our knowledge on the intracellular metabolic fluxes of the fungus in this complex process is limited, but appears highly relevant to better understand and rationally engineer the underlying metabolism. To quantify intracellular fluxes of growing and riboflavin producing A. gossypii, studies with different 13C tracers were embedded into a framework of experimental design, isotopic labeling analysis by MS and NMR techniques, and model-based data processing. The studies included the use 13C of yeast extract, a key component used in the process. Results During growth, the TCA cycle was found highly active, whereas the cells exhibited a low flux through gluconeogenesis as well as pentose phosphate pathway. Yeast extract was the main carbon donor for anabolism,  while vegetable oil selectively contributed to the proteinogenic amino acids glutamate, aspartate, and alanine. During the subsequent riboflavin biosynthetic phase, the carbon flux through the TCA cycle remained high. Regarding riboflavin formation, most of the vitamin’s carbon originated from rapeseed oil (81 ± 1%), however extracellular glycine and yeast extract also contributed with 9 ± 0% and 8 ± 0%, respectively. In addition, advanced yeast extract-based building blocks such as guanine and GTP were directly incorporated into the vitamin. Conclusion Intracellular carbon fluxes for growth and riboflavin production on vegetable oil provide the first flux insight into a  fungus on complex industrial medium. The knowledge gained therefrom is valuable for further strain and process improvement. Yeast extract, while being the main carbon source during growth, contributes valuable building blocks to the synthesis of vitamin B2. This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition

Document for adding numbered structures into PeakForest

This document is a standard operating procedure for the PeakForest infrastructure (peakforest.org)Its contains instructions for numbering molecules in relation with Nuclear magnetic resonance data