Objective THE AIM OF THE RESEARCH IS TO ELUCIDATE THE REGULATORY GENE AND THEIR EFFECTOR METABOLITES INVOLVED IN SWITCHING, FROM AEROBIC RESPIRATION TO FERMENTATION IN YEASTS. USE OF CONTINUOUS CULTURE/NMR METHODS TO ATTRIBUTE DETAILED PHENOTYPES TO EXISTING MUTATIONS, AS WELL AS OTHER MUTANT STRAINS WHICH WILL BE ISOLATED, WILL PROVIDE A VERY POWERFUL COMPLEMENT TO GENETIC STUDIES. THE COMBINED USE OF NMR SPECTROSCOPY WITH CONTINUOUS CULTURE IS A NEW NON-INVASIVE APPROACH TO STUDY YEAST PHYSIOLOGY. A reinterpretation of published data on the product fluxes and enzyme levels in S. cerevisiae, during growth on limiting glucose or ethanol, and analogous studies of the microbial physiology of Escherichia coli and of a thermophilic Bacillus resulted in the following conclusions. Genetic regulation is not involved in the switch from respiration to fermentation of sugars by yeast, but it is involved in the switch from fermentation of sugars to oxidation of ethanol.The yeast Saccharomyces cerevisiae is widely used as an efficient agent to ferment sugary liquids to alcoholic beverages and, more recently, to ethanol as a fuel for motor vehicles. The enzymes involved in the glycolysis account for 50% of the total cell protein.Research was carried out in order to identify the genes coding for factors which are required to enhance the synthesis of elevated levels of those enzymes which increase when yeast cells are exposed to fermentable sugars. This involved the isolation and characterization of those genes and their products which sense the availability of sugars, transmit a signal to the regulatory circuits which specifically activate transcription, translation and maturation of glycolytic enzymes. Mutants were isolated, genes cloned, sequenced, deleted and overexpressed. Direct mutagenesis in vitro was used to identify control regions of such genes and binding proteins. Overexpression of genes coding for different glycolytic enzymes was used to identify possible catalytic bottlenecks in glycolysis.Pyruvate decarboxylase was used as an indicator enzyme to study the regulation of glycolytic enzymes. A second structural gene PDC5 was found and shown to have great sequence similarity with PDC1 in the coding but not in the flanking 5 prime regions and 3 prime regions. PDC5 is only transcribed when PDC1 is deleted. PDC1 codes for all enzyme activity in wild type. When active, PDC5 promotes the formation of pyruvate decarboxylase at 80% the level of PDC1. Deletion analysis of the 5 prime region of PDC1 identified a sequence required for glucose induction of enzyme formation and a second region with repressing effects. Transcription activation factors were identified by protein deoxyribonucleic acid (DNA) binding assays. A new gene, PDC3, was identified as necessary for pyruvate decarboxylase formation at the posttranscriptional level. Induction of pyruvate decarboxylase requires the function of gene BYP1 involved in transducing a glucose triggered cyclic adenosine monophosphate (cAMP) signal.THE MAIN ROLE OF THIS PROJECT WILL BE TO DEVELOP METHODS, COMBINING NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY WITH CONTINUOUS CULTURE, TO DETERMINE THE DETAILED PHYSIOLOGICAL STATE OF YEAST STRAINS IN GLUCOSE LIMITED CHEMOSTATS. BY REGULATION THE DILUTION RATE IN SUCH CULTURES IT WOULD BE POSSIBLE TO CONTROL THE CARBON FLUX THROUGH FERMENTATIVE AND RESPIRATORY PATHWAYS. THE PROGRAMME WILL BE : - SET UP CONTINUOUS CULTURE OF WILD TYPE S. CEREVISAE, MONITOR KEY ENZYME LEVELS AND COMPARE LEVELS OF SOME METABOLITES IN FREEZE-CLAMPED SAMPLES BY CONVENTIONAL AND BY STATE NMR METHODS. - CONSTRUCT THE NMR BYPASS AND COMPARE IN VITRO MEASUREMENTS WITH "FREEZE-CLAMPED" VALUES. - EXPLORE TECHNIQUES TO SUPPRESS EXTRACELLULAR METABOLITE LEVELS FOR ETHANOL, PYRUVATE AND ACETATE TO ALLOW IN VIVO MONITORING OF INTRACELLULAR LEVELS. - USE 31 P AND 13 C ENRICHED "FED BATCH" EXPERIMENTS TO MEASURE FLUX THROUGH PATHWAYS. Fields of science natural sciencesbiological sciencesgeneticsDNAnatural scienceschemical sciencesorganic chemistryalcoholsnatural sciencesmathematicspure mathematicsarithmeticsprime numbersnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymesengineering and technologyindustrial biotechnologybioprocessing technologiesfermentation Programme(s) FP1-BAP - Multiannual research action programme (EEC) in the field of biotechnology (BAP), 1985-1989 Topic(s) Data not available Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE EU contribution No data Address South Kensington Campus LONDON United Kingdom See on map Total cost No data Participants (2) Sort alphabetically Sort by EU Contribution Expand all Collapse all Technische Hochschule Darmstadt Germany EU contribution No data Address Karolinenplatz, 5, 64289 Darmstadt See on map Total cost No data Trinity College Ireland EU contribution No data Address 2 Dublin See on map Total cost No data
