CLONING, ORGANIZATION AND FUNCTIONAL-ANALYSIS OF ILVA, ILVB AND ILVC GENES FROM CORYNEBACTERIUM-GLUTAMICUM

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of th is Gram+ bacterium and screened for clones carrying isoleucine biosynt hesis genes (ilv) by complementation of Escherichia coli mutants. Clon es complementing ilvA, ilvB, and ilvC were isolated. As based on the f unctional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy aci d synthase, and isomeroreductase, catalyzing three subsequent reaction s in Ile synthesis. Subcloning and transposon mutagenesis revealed tha t ilvB and ilvC reside on a 7-kb chromosomal fragment and that these g enes are transcribed in the same direction. A shuttle vector was const ructed to allow exonuclease treatment and assay subsets of plasmids fo r gene expression in the original C. glutamicum background. These cons tructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Souther n blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluste r was characterized. This fragment does not contain ilvA, demonstratin g the entirely different organization of the isoleucine biosynthesis g enes in C. glutamicum from that in entero bacteria.