Combining metabolic flux analysis with proteomics to shed light on the metabolic flexibility: the case of Desulfovibrio vulgaris Hildenborough.

Introduction: Desulfovibrio vulgaris Hildenborough is a gram-negative anaerobic bacterium belonging to the sulfate-reducing bacteria that exhibits highly versatile metabolism. By switching from one energy mode to another depending on nutrients availability in the environments" it plays a central role in shaping ecosystems. Despite intensive efforts to study D. vulgaris energy metabolism at the genomic, biochemical and ecological level, bioenergetics in this microorganism remain far from being fully understood. Alternatively, metabolic modeling is a powerful tool to understand bioenergetics. However, all the current models for D. vulgaris appeared to be not easily adaptable to various environmental conditions. Methods: To lift off these limitations, here we constructed a novel transparent and robust metabolic model to explain D. vulgaris bioenergetics by combining whole-cell proteomic analysis with modeling approaches (Flux Balance Analysis). Results: The iDvu71 model showed over 0.95 correlation with experimental data. Further simulations allowed a detailed description of D. vulgaris metabolism in various conditions of growth. Altogether, the simulations run in this study highlighted the sulfate-to-lactate consumption ratio as a pivotal factor in D. vulgaris energy metabolism. Discussion: In particular, the impact on the hydrogen/formate balance and biomass synthesis is discussed. Overall, this study provides a novel insight into D. vulgaris metabolic flexibility.

Corynebacterium glutamicum, a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro-aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild-type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L-1) corresponded to the lower kLa value of 5 h-1. Above 5 h-1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L-1. Taking into account this threshold, a fed-batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild-type strain was able to produce 93.6 g L-1 of succinate, which is one of the highest concentration reported in the literature.

Determination of reducing power and metal chelating ability of antioxidant peptides: revisited methods.

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way. After revision, it was then possible to determine an AERC indice (Ascorbate Equivalent Reducing Capacity) and a CECC (Carnosine Equivalent Chelating Capacity) or EECC (EDTA Equivalent Chelating Capacity) indice according to the standard chosen, by analogy to the TEAC indice (Trolox Equivalent Antioxidant Capacity) already used in many reported works to determine the free radical scavenging activity. Thus, the determination of these relative indices enables the comparison of antioxidative capacities obtained in various studies. The adaptation of these two tests to micro-scales and the calculation of AERC, EECC and CECC were performed on model peptides.

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures.

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r²=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.

Rapid screening of serum-free media for the growth of adherent Vero cells by using a small-scale and non-invasive tool.

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B(1) vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.