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Production of mannosylglycerate in an industrial strain using a recombinant mannosylglycerate synthase from Dehalococcoides

Mannosylglycerate is a compound frequently found in thermophilic and hyperthermophilic microorganisms where it accumulates concomitantly with salt and heat stress. A fundamental protective role in vivo has been ascribed to mannosylglycerate from the observation of the invariable association of this solute with high temperatures, occurring almost exclusively in thermophilic and hyperthermophilic microorganisms. The effect on biological structures has been demonstrated in vitro and mannosylglycerate was proven to be one of the best protein stabilizers against freeze-drying and thermal denaturation. Unfortunately, the utilization of mannosylglycerate in stabilization studies of biological structures such as enzymes, DNA and whole cells against heat, osmotic stress and dehydration, has been greatly restricted because its production to date, has been inefficient and expensive.

The culture media for growing natural mannosylglycerate-producing microorganisms are quite complex cost-limiting and the conditions of temperature and salt required by such organisms are highly corrosive to industrial fermentation equipment. Moreover, natural producers are not suited for industrial production, as they do not grow to high cell densities. In conclusion, it would be desirable that the large-scale production of MG for biotechnological studies and applications would be developed under mesophilic conditions and by simple and low cost methodologies. Saccharomyces cerevisiae was the organism of choice since it was genetically easy to manipulate and had been a valuable biotechnological tool for the production of important compounds of human utilization, as recently demonstrated, for example, with the complete biosynthesis of hydrocortisone.

The genes responsible for the synthesis of mannosylglycerate have only recently been identified and the corresponding enzymes have begun to be characterized. The pathway for synthesis of mannosylglycerate was described in the hyperthermophile Pyrococcus horikoshii and in the thermophile Thermus thermophilus. This pathway consists on the condensation of GDP-mannose and D-3-phosphoglycerate into an intermediate phosphorylated compound which is subsequently converted into mannosylglycerate.

The enzymes responsible for the process are encoded by two consecutive genes in hyperthermophilic and thermophilic prokaryotes. The analysis of the genome sequence of a mesophilic bacterium, Dehalococcoides ethenogenes, has shown that those sequences were also present in this organism, but as a fusion of both genes. We verified that the recombinant product of this bifunctional gene was capable of catalyzing the two final steps in mannosylglycerate biosynthesis. Cloning and transformation of this gene into the yeast Saccharomyces cerevisiae provided the organism with the ability to synthesize mannosylglycerate in vivo.

The ultimate objective of the present invention relates to processes for the production of high amounts of mannosylglycerate, a solute which properties have been demonstrated to be industrially applicable in the preservation of biomaterials, by transformed organisms that are suitable for industrial exploitation.

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CENTRO DE NEUROSCIENCIAS DE COIMBRA
Departamento de Zoologia, Universidade de Coimbra
3004-517 COIMBRA
Portugal
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