THIS RESEARCH WILL GIVE SOME CLUES ON THE MECHANISMS OF CARBON CATABOLITE REPRESSION IN YEASTS. THE AIM IS THEREFORE TO ELUCIDATE THE GLUCOSE CONTROL OF PYRUVATE DECARBOXYLASE STRUCTURAL GENE, PDC1, AND TWO GENES WITH REGULATORY FUNCTIONS, PDC2 AND PDC3.
TO CLONE THESE GENES IN A MULTICOPY YEAST PLASMID YEP13 TO ENHANCE THE CATALYTIC CAPACITY AT SPECIFIC REACTIONS IN GLYCOLYSIS IN ORDER TO DETERMINE POSSIBLE "BOTTLE NECKS" IN THE GLYCOLYTIC PATHWAY.
IT IS ALSO PROPOSED TO ELUCIDATE AN ALTERNATIVE PATHWAY IN GLYCOLYSIS WHICH CIRCUMVENTS THE PHOSPHOFRUCTOKINASE REACTION. MUTANTS LACKING PHOSPHOFRUCTOKINASE ACTIVITY ARE ABLE TO PRODUCE ETHANOL FROM GLUCOSE. THESE STUDIES ARE VERY IMPORTANT BECAUSE THEY WOULD FACILITATE THE UNDERSTANDING OF METABOLIC REGULATION IN YEASTS.
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 AIM OF THIS RESEARCH WILL BE TO DEPTH IN THE GENETIC EXPLORATION OF THE CATABOLIC PATHWAYS OF GLUCOSE IN THE YEAST SACCHAROMYCES CEREVISIAE. THE RESEARCH PROGRAMME WILL BE AS FOLLOWS :
YEAR 1 : CONSTRUCTION OF STRAINS CONTAINING GLYCOLYTIC ENZYME GENES ON MULTICOPY VECTOR YEP13. CLONING OF GENES BYP1, BYP2 AND BYP3. DESCRIPTION OF SUCH GENES. FIRST SERIES OF STUDIES USING THE CONTINUOUS CULTURE FACILITIES AND NMR ANALYSIS OF METABOLIC CONCENTRATION AT IMPERIAL COLLEGE.
YEAR 2 : CLONING OF GENES AFFECTING THE ACTIVITIES OF MORE THAN ONE GLYCOLYTIC ENZYME AND THE GENES SPECIFICALLY AFFECTING THE ACTIVITY OF PYRUVATE DECARBOXYLASE. SEQUENCING OF SELECTED CLONES. METABOLITE DETERMINATIONS IN MUTANTS, STRAINS WITH CLONED GENES ON MULTICOPY VECTORS AND GENE DISRUPTIONS USING THE CULTURING AND NMR FACILITIES AT IMPERIAL COLLEGE.
YEAR 3 : CONTINUATION OF CLONING AND SEQUENCING OF GENES CONTROLLING THE ACTIVITIES OF GLYCOLYTIC ENZYMES. FINAL ANALYSIS OF THE ENZYMES AND INTERMEDIATES IN THE BYP1-BYP2-BYP3 PATHWAY AND ANALYSIS OF MUTANT STRAINS.
Funding SchemeCSC - Cost-sharing contracts