Objective
THE PROPOSED RESEARCH WILL USE MODERN MOLECULAR GENETIC TECHNIQUES TO DEVELOP AND IMPROVE THE EXISTING GENETIC MANIPULATION SYSTEMS IN THE CORYNEBACTERIA.
THE MAJOR OBJECTIVE IS TO BE ABLE TO CLONE AND MANIPULATE KEY LIMITING STEPS IN INDUSTRIALLY IMPORTANT METABOLIC PATHWAYS IN THE CORYNEBACTERIA AND THEREBY CAUSE THE OVER-PRODUCTION OF HIGH METABOLITES (EG AMINO ACIDS).
LITTLE IS KNOWN ABOUT THESE BACTERIA AT A GENETIC LEVEL. A CLEAR KNOWLEDGE OF THE STRUCTURAL ORGANIZATION AND REGULATORY MECHANISMS IN THESE BACTERIA AT A MOLECULAR LEVEL, WOULD PERMIT THE GENETIC CONSTRUCTION OF SUPERIOR AMINO ACID PRODUCING STRAINS.
Genetic engineering techniques were developed in Corynebacter glutamicum and Bacillus lactofermentum to understand the mechanism and particularly the control of amino acid biosynthesis in these 2 bacteria. Research focussed on the development of vectors and plasmid transformation technology and the cloning and sequencing of genes in key amino acid biosynthetic pathways so as to identify control systems and devise protocols to deregulate them as a means to designing strains that overproduce amino acids. Genes involved in threonine and trytophan were chosen for study, since neither can currently be produced economically by fermentation.
New plasmid vectors, pULRS6 and pULRS8 were developed and transformed into corynebacteria (1E6 transformants/ug plasmid deoxyribonucleic acid (DNA) by the protoplast method. Plasmids were transformed into a range of coryneform bacteria by a novel electroporation system (1E7 ug plasmid DNA). A series of new promoter probe and bifunctional vectors were generated. Recombination deficient strains were studied. Several key amino acid biosynthetic genes were cloned and their gene organizations elucidated, viz the Thr biosynthetic gene hom-thrB; trp operon in B lactofermentum and in C glutamicum; aromatic pathway gene aroF. Promoters, attenuators and termination sequences of the trp operon of B lactofermentum were characterized by in vitro mutagenesis and the architecture of the trp attenuator and C glutamicum was determined by comparing DNA sequences of normal and depressed strains. These results are major advances in the molecular genetics of these 2 bacteria.
THE PROJECT WILL STUDY TWO VERY RELATED CORYNEBACTERIA: BREVIBACTERIUM LACTOFERMENTUM AND CORYNEBACTERIUM GLUTAMICUM WHICH ARE CURRENTLY USED IN THE AMINO ACID INDUSTRY.
THE WORK IS DIVIDED IN FOUR PARTS:
1.- IMPROVEMENT OF PLASMIDS VECTORS (CONSTRUCTION OF HIGHLY EFFICIENT VECTORS).
2.- RECOMBINATION IN CORYNEBACTERIA (PROTOPLAST FUSION)
3.- CLONING AND STRUCTURAL ANALYSIS OF GENES FOR AMINO ACID OIOSYNTHESIS IN CORYNEBACTERIA
4.- REGULATORY MECHANISMS OF CONTROL OF GENE EXPRESSION IN CIRYNEBACTERIA (IN VIVO AND IN VITRO)
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences molecular biology molecular genetics
- natural sciences biological sciences microbiology bacteriology
- natural sciences biological sciences genetics DNA
- medical and health sciences medical biotechnology genetic engineering
- natural sciences chemical sciences organic chemistry amines
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Coordinator
24071 LEON
Spain
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