THE POTENTIAL INTEREST OF BAKER'S YEAST SACCHAROMYCES CEREVISAE FOR NEW BIOTECHNOLOGICAL INDUSTRIES IS CONSIDERABLE IN SEVERAL FIELDS, SUCH AS PRODUCTION OF EUCARYOTIC PROTEINS OR METABOLITE OVERPRODUCTION. ITS USE AS A MODEL EXPERIMENTAL ORGANISM IN MOLECULAR BIOLOGY, ITS NON-PATHOGENICITY AND ITS ELABORATE GENETICS ARE WELL-KNOWN ADVANTAGES. HOWEVER, THE INDUSTRIAL PRODUCTION OF NEW BIOMOLECULES BY AEROBIC CULTIVATION OF YEAST REQUIRES BOTH A HIGH PRODUCTIVITY OF THE CARBON SOURCE AND A HIGH PRODUCT FORMATION RATE. THE PRESENT PROJECT IS CENTERED ON INVOLVING THESE PROBLEMS, THEORETICALLY AND EXPERIMENTALLY.
Iron sulphur proteins are involved as main catalysts in bioconversion processes (hydrogen evolution, nitrogen and carbon monoxide fixation, photosynthesis, respiration, etc). The metal active centres of different iron sulphur proteins (simple and complex, eg rubredoxin, ferredoxins and hydrogenase) were characterised and chemically modified, in order to produce new active structures with novel, and/or enhanced catalytic properties. A new concept of assisted inorganic synthesis by a protein template was developed for the synthesis of mixed metal clusters. The reactivity and stability of the newly formed catalysts and hydrogenase (free and immobilised) were tested by measuring hydrogen evolution/production, deuterium(2)/hydrogen(+) exchange (mass spectrometry) and hydrogenation activity. Another important goal was to develop an adequate system to test hydrogenation activity in a multiphase biocatalyst system with immobilised hydrogenase or bacterial cells.
A biochemical and genetic study was made of the mechanisms involved in amino acid overproduction and export in the yeast Saccharomyces cerevisiae, using aromatic amino acids as the experimental system. The metabolic bottlenecks opposing maximum product formation in deregulated and/or transformed strains were investigated. Mass culture conditions were analysed and a partially automatic laboratory fermentation system was developed ensuring the most efficient product formation by the developed strains.
Mutations favouring overproduction and excretion of aromatic amino acids were isolated. Strains bearing 1 or several of these mutations were constructed and their enzymatic, growth and excretory properties examined. Certain implicated genes were cloned and sequenced, making it possible to manipulate them in vitro. This is the case of the prephenate dehydrogenase gene, which was shown to play a decisive role in channelling the metabolic flow into tyrosine or phenylalanine production.
A sophisticated automated fermentation control system, adaptable to genetically modified yeast strains, was developed and is being improved constantly. The studies concerning growth inhibition of overproducing strains revealed the role of excess acetate formation and the dysfunction of the aromatic transamination reactions.
THE PROJECT AIMS TO IMPROVE THE OPTIMIZATION OF THE GROWTH IN FERMENTORS OF SACCHAROMYCES CEREVISAE STRAINS GENETICALLY MANIPULATED FOR THE PRODUCTION OF INDUSTRIALLY INTERESTING METABOLITES.
THE RESEARCH PROGRAMME WILL TAKE THE FOLLOWING STEPS :
1. KINETICS PROGRAMME AND PHYSIOLOGY OF YEAST GROWTH.
1.1 KINETICS AND PHYSIOLOGY IN BATCH CULTURES.
1.2 KINETICS AND PHYSIOLOGY IN CONTINUOUS AND FEED BATCH CULTURES.
2. OPTIMIZATION AND AUTOMATION OF THE YEAST GROWTH.
3. OPTIMIZATION AND AUTOMATION OF THE PRODUCTION OF "INTERESTING METABOLITES".
Funding SchemeCSC - Cost-sharing contracts