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Physiology and molecular genetics of amino acid production and secretion by corynebacteria : flux of intermediates and feedback control mechanisms

Objective

The main objective of this collaborative research proposal is to develop the molecular genetics of corynebacteria in order to understand the physiology of this group of microorganisms which are used industrially for the production of amino acids, flavour enhancing agents and in bioconversions of steroids, vitamins and a variety of other compounds.
An array of basic techniques for genetic manipulation of amino acid producing corynebacteria and related Gram-positive organisms have been developed. New improvements for gene transfer technology have lead to cloning several genes involved in intermediary metabolism of corynebacteria such as genes that provide precursors for amino acid biosynthesis and also genes directly involved in the production of several amino acids (tryptopahn, threonine, lysine, etc). New phage-derived tools are also being implemented by genetic modification of corynebacteria.
Techniques of pulse field electrophoresis have been sucessfully used for isolating large fragments of the genome of several corynebacteria. In combination with cosmid gene libraries, this technique is being applied for construction of ordered encyclopedias of the genome of B lactofermentum and C glutamicum.
Characterization of corynebacterial promoters and regulatory sequences and directed in vitro mutagenesis of those sequences has led to construction of strong expression systems in which production of amino acids is deregulated.
Corynebacteria are widely used in the production of amino acids and flavour enhancing agents, in bioconversions of steroids and intermediates in vitamin C biosynthesis and in the cheese industry. Some corynebacteria are also important plant pathogens.

The aim of this collaborative research between five groups already established in the field of amino acid production by corynebacteria is to further develop the molecular genetics of corynebacteria in order to understand the physiology of this group of microorganisms which are used industrially for production of amino acids in Europe, in Japan and the USA.

The main objectives of the collaborative research project are:

development of phage derived genetic tools for corynebacteria, particularly construction of cosmid vectors, transduction systems and characterization of the integration sequences of temperate bacteriophages;
characterization of DNA sequences required for plasmid replication and plasmid stability in liquid cultures (fermenters) and construction of integrative vectors;
development of genetic methods for the analysis and manipulation of corynebacteria genes relevant for amino acid production (gene disruption and gene replacement, transposon mutagenesis, construction of physical maps and cosmid libraries);
regulation of the tricarboxylic acid (TCA) cycle enzymes in order to increase the availability of the glycolysis and TCA cycle derived precursors for amino acid biosynthesis;
characterization of promoters of the amino acid biosynthetic genes and feedback control mechanisms of the amino acid biosynthetic pathways in order to remove bottlenecks in the pathways for overproduction of amino acids.

The project is an integrated approach (genetics and physiology) to study amino acid biosynthesis in corynebacteria. The results of this research will also be useful in other applications of corynebacteria, eg in biotransformations and in the interaction of pathogenic corynebacteria with plants.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Universidad de León
Address
S/n,campus De Vegazana
24071 León
Spain

Participants (4)

Forschungszentrum Jülich GmbH
Germany
Address
Wihelm-johnen-straße
52405 Jülich
UNIVERSITY OF BIELEFELD
Germany
Address
Universitaetstrasse 25
33501 Bielefeld
University College Galway
Ireland
Address
University Road
90 Galway
Université de Rennes I
France
Address
Avenue Du Général Leclerc
35042 Rennes