Marc Perez

Use of a continuous multistage bioreactor to mimic winemaking fermentation.

Continuous fermentation set-ups are of great interest for studying the physiology of microorganisms. In winemaking conditions, yeasts go through a growth phase and a stationary phase during which more than half of the sugar is fermented. A comprehensive study of wine-yeast physiology must therefore include yeasts in a non-growing phase. This condition is impossible to achieve within a chemostat, which led us to design a multi-stage fermentation device. In this study, we evaluated the ability of such a device to reproduce, in a series of steady states, the conditions of batch fermentation. Two-stage and four-stage fermentations were carried out with two different strains of Saccharomyces cerevisiae. The main characteristics of the fermentation process (biomass growth, by-product content of the medium) were compared with those observed in batch mode at the same stage of fermentation, which was defined by glucose uptake. The four-stage configuration showed a better ability to reproduce batch fermentation characteristics than the two-stage set-up. It also allowed to uncouple the variations of environmental parameters and proved to be a promising tool to gain new insights into yeast metabolism during alcoholic fermentation.

Metabolic Responses of Saccharomyces cerevisiae to Valine and Ammonium Pulses during Four-Stage Continuous Wine Fermentations

ABSTRACT Nitrogen supplementation, which is widely used in winemaking to improve fermentation kinetics, also affects the products of fermentation, including volatile compounds. However, the mechanisms underlying the metabolic response of yeast to nitrogen additions remain unclear. We studied the consequences for Saccharomyces cerevisiae metabolism of valine and ammonium pulses during the stationary phase of four-stage continuous fermentation (FSCF). This culture technique provides cells at steady state similar to that of the stationary phase of batch wine fermentation. Thus, the FSCF device is an appropriate and reliable tool for individual analysis of the metabolic rerouting associated with nutrient additions, in isolation from the continuous evolution of the environment in batch processes. Nitrogen additions, irrespective of the nitrogen-containing compound added, substantially modified the formation of fermentation metabolites, including glycerol, succinate, isoamyl alcohol, propanol, and ethyl esters. This flux redistribution, fulfilling the requirements for precursors of amino acids, was consistent with increased protein synthesis resulting from increased nitrogen availability. Valine pulses, less efficient than ammonium addition in increasing the fermentation rate, were followed by a massive conversion of this amino acid in isobutanol and isobutyl acetate through the Ehrlich pathway. However, additional routes were involved in valine assimilation when added in stationary phase. Overall, we found that particular metabolic changes may be triggered according to the nature of the amino acid supplied, in addition to the common response. Both these shared and specific modifications should be considered when designing strategies to modulate the production of volatile compounds, a current challenge for winemakers.

Efficient therapy for refractory Pompe disease by mannose 6-phosphate analogue grafting on acid α-glucosidase

Abstract Pompe disease is a rare disorder due to deficiency of the acid &agr;‐glucosidase (GAA) treated by enzyme replacement therapy. The present authorized treatment with rhGAA, the recombinant human enzyme, provides an important benefit in the infantile onset; however, the juvenile and adult forms of the disease corresponding to > 80% of the patients are less responsive to this treatment. This resistance has been mainly attributed to an insufficiency of mannose 6‐phosphate residues in rhGAA to address lysosomes through the cation‐independent mannose 6‐phosphate receptor (CI‐M6PR). As yet, several attempts to improve the enzyme delivery by increasing the number of mannose 6‐phosphate on the enzyme were poorly effective on the late onset form of the disease. Here, we show that chemical conjugation of a synthetic analogue of the mannose 6‐phosphate, named AMFA, onto rhGAA improves the affinity for CI‐M6PR and the uptake of the enzyme in fibroblasts and myoblasts of adult Pompe patients. More importantly, only the conjugated rhGAA‐AMFA was effective in aged Pompe mice when compared to rhGAA. Weekly treatment with 5–20 mg·kg− 1 rhGAA‐AMFA provided major improvements of the motor function and of the myofiber structure, whereas rhGAA was inactive. Finally, AMFA addition did not induce supplementary immune response to the enzyme. This modified enzyme, displaying a muscle recovery in aged Pompe mice that was never attained before, could be considered as a potential therapy for the late onset Pompe disease. Graphical abstract Figure. No Caption available.

Design of Potent Mannose 6-Phosphate Analogues for the Functionalization of Lysosomal Enzymes To Improve the Treatment of Pompe Disease

Improving therapeutics delivery in enzyme replacement therapy (ERT) for lysosomal storage disorders is a challenge. Herein, we present the synthesis of novel analogues of mannose 6-phosphate (M6P), known as AMFAs and functionalized at the anomeric position for enzyme grafting. AMFAs are non-phosphate serum-resistant derivatives that efficiently bind the cation-independent mannose 6-phosphate receptor (CI-M6PR), which is the main pathway to address enzymes to lysosomes. One of the AMFAs was used to improve the treatment of the lysosomal myopathy Pompe disease, in which acid α-glucosidase (GAA) is defective. AMFA grafting on a M6P-free recombinant GAA led to a higher uptake of the GAA in adult Pompe fibroblasts in culture as compared to Myozyme, the M6P recombinant GAA. Moreover, the treatment of Pompe adult mice with the AMFA-grafted recombinant enzyme led to a remarkable improvement, even at low doses, in muscle functionality and regeneration, whereas Myozyme had limited efficacy.

Control of a Multi-Stage Continuous Fermentor for the study of the wine fermentation

Yeasts play a central role in the wine making process. To study the yeasts in a stable environment and physiological state, a Multi-Stage Continuous Fermentor (MSCF) has been designed that mimics steps of the batch fermentation process. In this paper, the problem of the control of the sugar concentrations in each of the four reactors of the MSCF is considered. The cascade structure of the device leads to a constraint on the input flow rates (the control variables). A control strategy based on a linearizing control law coupled with a state observer and an anti windup component is proposed and finally implemented on the experimental process.

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

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.

Control of a class of nonlinear cascade systems with input-dependent saturations

In the present paper, we propose a control strategy for the stabilization of a class of nonlinear cascade systems with input-dependent saturation. The saturation constraint considered is a constraint of order: the control input of one subsystem has to be smaller than the control input of the preceding subsystem of the cascade structure. This class of systems include interconnected reactors device such as multi-stage continuous fermentors. The control law is designed on the basis of a specific Lyapunov function previously introduced by R. Antonelli and A. Astolfi in [1]. It guarantees the stability of the closed-loop system, the convergence of the state variable to the set-point and the fulfillment of the saturation constraint all along the trajectories of the closed-loop system. The approach is applied, in numerical simulation, to a concrete example of a series of interconnected continuous bioreactors.

Interest in Online Higher Alcohol and Ester Determinations during Winemaking Fermentations

To access the kinetics of synthesis of higher alcohols and esters during the alcoholic fermentation process, we developed an original online gas chromatography device. The high frequency measurement made it possible, for the first time, to calculate rates and specific rates of production. In parallel, it was possible to perform dynamic gas–liquid balances. Losses highly depended on the studied compound. They were negligible for the higher alcohols but high for the esters, even at moderate temperatures.