THE PROPOSED RESEARCH WILL USE MODERN MOLECULAR 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 OVERPRODUCTION OF HIGH METABOLITES (E.G. 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.
THIS JOINT RESEARCH WILL USE MODERN GENETIC TECHNIQUES TO DEVELOP AND IMPROVE THE GENETIC MANIPULATION OF THE CORYNEBACTERIUM GROUP OF BACTERIA WHICH ARE CURRENTLY USED OR POTENTIALLY USEFUL IN THE INDUSTRIAL PRODUCTION OF AMINO ACIDS, NUCLEOTIDES AND OTHER USEFUL METABOLITES.
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 OVERPRODUCTION OF THESE METABOLITES.
THE RESEARCH STRATEGY AND METHODOLOGY IS DIVIDED INTO FOUR SECTORS :
1. IMPROVEMENT OF PLASMID VECTORS FOR C. GLUTAMICUM.
2. PLASMID DELIVERY SYSTEMS FOR CORYNEBACTERIUM GLUTAMICUM.
3. TRANSPOSON INTRODUCTION.
4. AMINO ACID PATHWAY ANALYSIS BY SITE-DIRECTED MUTAGENESIS IN CORYNEBACTERIUM AND BREVIBACYERIUM.
Funding SchemeCSC - Cost-sharing contracts