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ENVIRONMENTAL CONTROL OF METABOLIC FLUXES AS A BASIS FOR BIOTECHNOLOGICAL PROCESSES

Objective

THE AUTHORS HAVE DEVELOPED TECHNIQUES FOR THE ISOLATION OF ENDOSPORE FORMING ORGANISMS BELONGING TO THE GENUS BACILLUS. AMONG THESE WAS AN ISOLATE ABLE TO GROW OPTIMALLY AT 55C WITH A DOUBLING TIME OF 75MIN IN MEDIA CONTAINING METHANOL AS THE SOLE CARBON AND ENERGY SOURCE. THE METHODOLOGY WILL INVOLVE ENZYME ANALYSIS, BATCH AND CHEMOSTAT CULTIVATION UNDER A VARIETY OF CONDITIONS, INCLUDING THE USE OF MIXED SUBSTRATES AND THE APPLICATION OF GENETIC TECHNIQUES TO STUDY THE MOLECULAR BIOLOGY OF THE REGULATION OF GENE EXPRESSION.
Novel thermotolerant methylotrophic bacilli were isolated in order to elucidate their pathways(s) of methanol metabolism and mode(s) of regulation. By means of genetic and physiological manipulation, metabolism was guided towards an overproduction of important biochemicals. Environmental parameters were independently manipulated using chemostat culture techniques and specific gene functions disrupted to determine where precisely the regulation of metabolite flux resides.

Thermophilic bacilli capable of growth on methanol as the sole carbon substrate were isolated in pure culture and characterized metabolically. All strains possessed an nicotinamide adenine dinucleotide (NAD) linked methanol (alcohol) dehydrogenase (MDH) and generally grew on methanol with a very high efficiency. MDH had a low affinity for methanol but, along with reduced nicotinamide adenine dinucleotide (NADH) oxidase and hexulose phosphate synthase, was synthesized at very high levels. Similarly, Bacillus stearothermophilus synthesized glycerol kinase at extremely high levels and, when growing in glycerol-limited chemostat culture, had up to 40% (weight for weight) of its total protein as this 1 enzyme. Clearly, these organisms possess highly efficient expression systems. Studies of glucose metabolism in yeast revealed novel information on the role of cyclic adenosine monophosphate (cAMP) and protein phosphorylation in the regulation of glucose uptake and fermentation.
THIS PROPOSAL AIMS TO INVESTIGATE THE METABOLIC BEHAVIOUR OF SPECIES OF THE GENUS BACILLUS. MEMBERS OF THIS GENUS ARE CURRENTLY BEING USED IN A NUMBER OF INDUSTRIAL PROCESSES. IT IS PROPOSED TO EXAMINE, IN A FEW SELECTED BACTERIAL SPECIES, THE REGULATION OF CARBON SUBSTRATE METABOLISM, ITS BIOENERGETIC CONSEQUENCES AND THE CARBON FLOW INTO METABOLITES WITH AN INDUSTRIAL POTENTIAL.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

RIJKSUNIVERSITEIT GRONINGEN
Address
Lab. Voor Microbiologie Kerklaan 30
Groningen
Netherlands