Objectif
Extremophiles have tremendous potential in biotechnology because of their ability to grow under extreme conditions and to produce unique stable products with high technological value. By integrating the activities of 39 expert laboratories we were able to isolate and characterize a variety of novel microorganisms that can survive under extreme conditions (extremes of temperature, pH and high salt concentrations). Using an interdisciplinary research activities involving microbiologists, biochemists, chemists and engineers cell yields up to 132 g/l (cell dry weight) were obtained. This was a prerequisite for the isolation and production of various products such as enzymes, lipids and organic solutes that are of interest for various industrial applications. The structure-function relationship of enzymes from extremophiles such as glutamate dehydrogenase and hydrolases were studied. The analysis of the three dimensional structure of a number of enzymes from extremophiles lead to the elucidation of the principle of thermostability and salt tolerance. Novel compatible solutes were identified in extremophiles and were found to have a positive effect on the stabilization of enzymes. Gene regulation and control could be studied in detail in many representatives of extremophiles and the mechanisms involved in energy transduction were elucidated. Due to the interdisciplinarity and collaborative interaction of the project leaders and their coworkers, these major scientific breakthroughs have been achieved. This is documented by 103 joint publications (270 in total) in international scientific journals and the exchange of students and scientists.
Isolation and development of fermentation technology
Laser microscope (optical tweezers) was successfully applied for the isolation of Archaea and Bacteria. By applying this fast technique five different thermoacidophilic strains were isolated from smouldering slag heaps in Sweden. Gram-negative sulphur oxidizing alkaliphilic bacteria and Actinomycetes were isolated from soda lake samples from Siberia and East Africa. Furthermore, microorganisms were isolated from Lake Bogoria that are able to grow at high temperatures (60-80(C) and pH values (9.5-11.0). The MEE (Multilocus Enzyme Electrophoresis) method has been developed and found to be both rapid and reliable for the classification of thermophiles. The phylogentic position of extremely halophilic archaea isolated from the Dead Sea was also determined. Cultivation of hyperthermophiles was performed in dialysis batch, gas-lift bioreactors as well as by complete cell recycling with cross-flow filtration techniques. By fermenting the organism in a dialysis membrane bioreactor a dramatic increase in the maximal cell density (3.5E10 cells/ml, O.D. 6.0) could be observed. The influence of various medium components such as sugars, amino acids and vitamins on growth of the hyperthermophiles Pyrococcus furiosus, Pyrococcus abyssi and Aquifex pyrophilus was investigated. Cheaper industrial grade substrates were identified and over 1 kg of pyrococcal cell paste was produced in gas lift reactors. The cultivation of the hyperthermophile Pyrococcus furiosus, the thermoacidophile Sulfolobus shibatae and the halophile Marinococcus M52 in dialysis membrane reactors resulted in cell yields of 2.6 114 and 132 g/l (cell dry weight), respectively. These results demonstrate that cultivation of extremophiles to high cell densities can be achieved if modern fermentation technology is employed.
Unique enzymes from extremophiles
Amylase from the hyperthermophile Pyrococcus woesei, which is an interesting enzymes for the starch industry, was purified, characterized and its gene was cloned in the mesophilic hosts Escherichia coli and Bacillus subtilis. The pullulanase from the same Archaeon was crystallized and used to collect a native and two derivative intensity data sets by X-ray diffraction. The glutamate dehydrogenase from this model organism was also crystallized and studied in detail. The comparative analysis of the structures of pyrococcal and clostridial GluDH revealed a major role for ion pair networks in maintaining enzyme stability. Crystals of Alcohol dehydrogenase from Sulfolobus solfataricus were prepared for further analysis. The beta-glycosidase has been recently crystallized and the 3D structure has been resolved at 2.6 A. In addition the DNA polymerase, an interesting enzyme for PCR technology, from Sulfolobus solfataricus was cloned and expressed in E. coli.
In order to understand the basis of protein thermostability the hexameric GluDH from Pyrococcus furiosus and glutamate dehydrogenase have been investigated. The refolding pathway was analyzed, the assembly intermediates were identified and their formation rate constants were calculated. The detailed characterzation of archaeal-bacterial hybrid showed that thermal and chemical stability may be uncoupled. Chaperonin from Sulfolobus solfataricus has been shown to promote correct folding of several thermophilic and mesophilic enzymes from their chemical denatured state by preventing aggregation of refolded intermediates or native enzymes. The two enzymes adenylate kinase and cytosolic pyrophosphatase from Sulfolobus acidocaldarius have been investigated. Both genes were successfully expressed in E. coli and the proteins were crystallized.
The thermostable xylanase, which has application potential in biopulping, from Rhodothermus marinus has been successfully synthesized in E. coli. Other polymer degrading enzymes such as protease and amylase were studied from newly isolated psychrophylic microbes. Both enzymes have been purified and characterized. Alkali stable and thermostable amylase and pullulanase have been identified from a newly isolated thermoalkaliphile which represent a new taxon Thermopallium natronophilum. In addition, the most thermostable cellulase known so far has been purified from Rhodothermus marinus. The P. furiosus genes for a novel endoglucanase and several alcohol dehydrogenases have been determined and functionally expressed. Also the serine protease (pyrolysin) has been purified and the pls gene coding for this pre-pro-protein has been completely sequenced. Two DNA topoisomerases were purified from P. furiosus. Finally, the novel ADP dependent kinase has been purified and characterized.
Metabolism of carbohydrates and production of low molecular weight compounds
Glucose metabolism was investigated in seven strains of hyperthermophilic archaea and bacteria using 13C-labelled substrates. Thermococcus tenax and Thermotoga maritima show considerable contributions to both the Embden-Meyerhof and the Entner-Doudoroff pathways, whereas Thermococcus and Pyrococcus strains show 100% Embden-Meyerhof and Sulfolobus strains use exclusively the Entner-Doudoroff pathway. The novel archaeal enzyme involved in acetate formation and ATP synthesis (acetyl-CoA synthetase) was purified from Pyrococcus furiosus and characterized. Several strains of Thermotogales were screened for the presence of solutes (low molecular weight organic compounds). A novel compound derived from di-myo-inositol-phosphate was found in Thermotoga maritima and characterized by NMR. Novel organic solutes (di-glycerol-phosphate, galactosyl-5-hydroxylysine and mannosylglycerate) were identified in archaea and their influence as an enzyme protector against heating and freeze drying was shown. Furthermore, six novel hopanoids, bacteriohopanetetrol glycosides possessing a glycuronic acid moiety, were isolated from a thermophilic Cyanobacterium, Synechococcus sp.
Gene regulation and control
In this project it was possible to establish a cell-free transcription system for the hyperthermophile working at 90(C. Two pyrococcal transcription factors were discovered and were found to be related in their structure and function to eucaryotic transcription factors TBP and TFIIB. New plasmids were isolated from Thermococcales and a naturally positively supercoiled plasmid was identified for the first time in vivo. Furthermore, the DNA gyrase from Thermotoga maritima was sequenced and lead to the discovery of a new family of Topo II in hyperthermophilic archaea. An efficient transformation procedure was developed for Pyrococcus which was followed by the development of a shuttle vector between E. coli and P. furiosus. It has been also demonstrated that both the archaeal introns and their genetically engineered derivatives are mobile between hyperthermophiles. Saccharomyces cervisiae was proven to be a particularly adequate host for the expression of the hyperthermophilic P. furiosus OTCase whereas E. coli is not, due to partial proteolysis. Furthermore, a suitable shuttle vector was constructed for moderate halophiles and conjugation was demonstrated. Native promotors from various moderate halophiles were isolated and characterized. DNA fragments influencing osmoregulation were isolated. It has been shown that the differential streptomycin transport efficiency in haloalkaliphilic bacteria was dependent on the ionic strength of the medium.
Membranes, membrane proteins and bioenergetics of Extremophiles
The investigations on the bioenergetics of extremophiles have shown that a correlation exists between the optimum growth temperature and the proton permeability of the membrane. To study this phenomenon in further detail liposomes were prepared from lipids derived from various extremophiles grown at various temperatures. Energy transduction was studied in Picrophilus oshimae, a thermoacidophilic archaea that grows optimally at pH 0.7 and 60(C. This archaeon maintains its intracellular pH at 5.0 with the external pH of 0.7. Membranes of this organism were found to be equipped with a low proton permeability that allows proton-linked energy-transduction. It has been also shown that the energy-transduction in the newly isolated anaerobic thermophile strain LBS3 was linked to sodium-cycling. The electron transport complexes (Rieske-FeS proteins, succinate dehydrogenase, terminal heme-copper oxidases) involved in respiratory energy transduction were investigated. Two Rieske-FeS proteins were discovered in Sulfolobus acidocaldarius and their genes were sequenced. Proton pumping by a Q-cycle was found in the terminal oxidase complex of Sulfolobus and two operons encoding the unusual terminal oxidase from Desulfurolobus ambivalens were completely resolved. In addition, Rhodothermus marinus respiratory chain was deeply studied by chemical, spectroscopic and molecular biology tools.
Champ scientifique (EuroSciVoc)
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CORDIS classe les projets avec EuroSciVoc, une taxonomie multilingue des domaines scientifiques, grâce à un processus semi-automatique basé sur des techniques TLN. Voir: Le vocabulaire scientifique européen.
- sciences naturelles sciences biologiques microbiologie bactériologie
- sciences naturelles sciences biologiques génétique ADN
- sciences naturelles sciences biologiques biochimie biomolécule lipides
- sciences naturelles sciences chimiques chimie organique alcool
- sciences naturelles sciences biologiques biochimie biomolécule protéines enzyme
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