Publication

Production of a thermostable fumarase in Saccharomyces cerevisiae for the bioconversion of fumarate to L-malate

Coralie Signorell
2011
Projet étudiant
Résumé

The gene encoding fumarase (fum) from Thermus thermophilus was expressed in yeast Saccharomyces cerevisiae. The recombinant cells were heated at 70°C to inactivate indigenous enzymes and used for the bioconversion of fumaric acid to L-malic acid. By heating the host cells at 70°C, substrate is able to go across the heat-damaged membrane of the microorganism and a desired product can be formed. This new concept, called Synthetic Metabolic Engineering (SME), has already been applied successfully in Escherichia coli. Unfortunately, E. coli membrane is weakened too much during the heat treatment and enzyme leakage appears. The surface structure of yeast is more rigid than that of E. coli and this might be taken as an advantage for application of SME. When continuous or repeated-batch reaction is carried out, enzyme leakage becomes a major drawback. It is assumed that yeast cells could overcome this problem by retaining more enzymes in the cell during and after the heat treatment. In order to prove this hypothesis, a thermophilic fumarase (FUM) was over-expressed in two hosts, S. cerevisiae as well as E. coli. fum was first modified to be over-expressed in yeast cells and FUM was successfully produced in yeasts. Optimization of SME techniques was carried out for yeast cells. Then, enzyme activity and enzyme leakage was investigated for both strains. E. coli showed high level of FUM expression, though considerable amount of enzyme leaked to supernatant. On the other hand, even though the level of FUM expression in S. cerevisiae was low, yeast cells overcome leakage problem and are re-usable. This study showed the first trial of SME in yeast cells and possibility of utilization of yeast as a host strain for SME.

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Concepts associés (32)
Levure
Une levure est un champignon unicellulaire apte à provoquer la fermentation des matières organiques animales ou végétales. Les levures sont employées pour la fabrication du vin, de la bière, des alcools industriels, des pâtes levées, des antibiotiques et d'exhausteurs de goût (les extraits de levure peuvent servir comme agents de sapidité). Le terme « levure » sans spécification peut avoir un emploi générique ou spécifique, dont on vient de donner deux définitions, l'une au singulier et l'autre au pluriel, toutes les deux renvoyant à une classe large d'espèces que la définition spécifie.
Acide malique
L'acide malique est un acide dicarboxylique alpha-hydroxylé de formule . Il existe sous la forme de deux diastéréoisomères, les énantiomères L et D ; seul l'acide L-malique est présent dans le milieu naturel. La base conjuguée de l'acide malique et ses sels sont appelés malates. L'anion L-malate est un intermédiaire du cycle de Krebs, l'une des voies métaboliques majeures de respiration cellulaire chez presque tous les êtres vivants, et intervient dans le cycle de Calvin, qui entre dans le cadre de la photosynthèse.
Escherichia coli
Escherichia coli, en abrégée E. coli, est une bactérie intestinale des organismes à sang chaud, Gram négatif, du genre Escherichia, en forme de bâtonnet. E. coli est une bactérie aero-anaerobie facultative, appartenant au groupe des colibacilles, très commune chez l'être humain. E. coli constitue, avec d'autres bactéries anaérobies facultatives, 0,1% du microbiote intestinal. Découverte en 1885, par le pédiatre et bactériologiste austro-allemand Theodor Escherich, dans des selles de chèvres, c'est un coliforme fécal généralement commensal.
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