Risultati finali
We have established an industrial production procedure for mannosylglycerate (MG) and mannosylglyceramide (MGA) and tested the usefullness of these compounds in different applications. As a result two patents have already been filed for the use of MG and MGA in cosmetic formulations and as adjuvants in vaccines.
These patents are based in the superior capabilities of these two solutes to serve as moisturising agents and as protein protective agents against thermal denaturation or/and repetitive use.
The accumulation of low-molecular mass organic solutes such as, trehalose, polyols or ectoines, is a prerequisite for osmotic adjustment of many mesophilic microorganisms. However, very unusual solutes namely, di-myo-inositol-phosphate, di-mannosyl-di-myo-inositol-phosphate, diglycerol phosphate, mannosylglycerate, and mannosylglyceramide, have been identified in thermophilic and hyperthermophilic microorganisms and the intracellular content of these solutes increases in response to stress conditions, such as high osmolarity or high temperature.
Mannosylglycerate and diglycerol phosphate have been studied to a greater extent and have been shown to protect enzymes and proteins in vitro better than compatible solutes from mesophiles [1-3]. Moreover, the application of compatible solutes from thermophilic or hyperthermophilic organisms as stabilising agents of biomaterials has been disclosed in several patent applications [4-6].
We have discovered a novel compatible solute in the hyperthermophilic bacterium Aquifex pyrophilus, and in the hyperthermophilic archaeon Archaeoglobus fulgidus. Both organisms share, approximately, the same growth conditions (optimal growth around 80oC and 2% NaCl). When subjected to a combination of salt and temperature stresses both organisms accumulate the novel compound.
In the case of Aq. pyrophilus this accumulation, as a response to a combination of temperature and salt stress, can be massive reaching more than 2 μmol/mg of protein, and becoming the major accumulating solute. After extraction, purification, and full spectroscopic characterization by Nuclear Magnetic Resonance, we have determined the molecular structure of this, to date, unidentified compound as 1-phosphoglycerol-1-myo-inositol or glycerophosphoinositol (GPI).
It is interesting to note that the molecular structure of this compatible solute resembles a chimera between di-myo-inositol-1,1 -phosphate (DIP) and di-glycerol-1,1-phosphate (DGP). While DIP is a widely distributed solute among hyperthermophiles responding strongly to supraoptimal growth temperatures [7], DGP is a strong protein thermal stabilizer [3].
Because of the thermal protection abilities of DGP and DIP, industrial utilizations of both solutes are protected under European patent applications [5-6]. The thermophilic origin of the novel solute, its pattern of accumulation in response to combined temperature and salt stress, and its structural resemblance to DIP and DGP leads us to propose that this novel solute has stabilising properties as good or superior to those already demonstrated for DIP and DGP. In this respect, it can serve as a stabiliser in various commercial, industrial, medical, pharmaceutical, diagnostic, cosmetic, or academic research applications.
Although, glycerophosphoinositol had generically been previously envisioned as a possible cryopreservative in long term storage of tissues from animal or vegetable origin [8], its use as a highly efficient protector of enzymes, proteins, lipid vesicles or microbial cell lines against heat salt stress, desiccation, transport or routine usage, had never been disclosed.
The enhanced protein stability rendered by certain low-molecular mass organic solutes allows enzymes to function under more severe conditions of temperature, pressure, ionic strength, pH, presence of detergents or organic solvents. One of the priorities of modern biotechnology is to obtain stable enzymes or agents that stabilise those enzymes against thermal or chemical denaturation.
The ability of some compatible solutes to stabilise enzymes is, therefore, of great importance to modern biotechnology. This point is obviously extended to all proteins that are used or can be used in processes where their stability is an issue, since all proteins either with or without enzymatic activity share the same overall basic elements of structure and may be protected against denaturation or inactivation through the same general mechanisms or processes.
It must also be stressed that compatible solutes protect proteins, cell membranes, and lipossomes from the deleterious effects of desiccation, and possess strong moistening properties. The preservation of desiccated or lyophilized cell components and biomaterials has many applications in medicine, pharmaceutical industry, cosmetic industry, food industry, and scientific research. In spite of the great importance of desiccation and freezing in the conservation of biological samples, denaturation of proteins, disruption of lipossomes, or degradation of nucleic acids inevitably takes place during utilization or transport, and could be prevented or diminished by the use of low molecular mass stabilisers.
Also, the stability of nucleic acid molecules, like DNA, or RNA, can be improved by the addition of compatible solutes from hyperthermophiles, as described for ectoines [9], and their use in several applications in medicine, pharmaceutical industry, or scientific research can be envisioned.
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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