Construction of l -Isoleucine Overproducing Strains of Corynebacterium glutamicum

Abstract

is used for the industrial production of the amino acids l-glutamate (1×106 tons/year) and l-lysine (300×103 tons/year). The classical approach to obtain amino acid overproducing strains of C. glutamicum was mutagenesis and then a selection of mutants. In the past 10 years the genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for constructing microbial strains with desired genotypes. To obtain l-isoleucine overproducing strains of C. glutamicum we therefore studied the l-isoleucine biosynthesis by overexpression of the various corresponding genes. To enable a flux increase in recombinant strains all genes specific for l-threonine and l-isoleucine biosynthesis were cloned from this bacterium. We demonstratet that amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enable the channeling of the carbon flow from the intermediate l-aspartate semialdehyde towards homoserine, resulting in an accumulation of l-threonine. To obtain effective l-isoleucine overproduction a deregulated threonine dehydratase was overexpressed in l-threonine producing strains of C. glutamicum. In this way the l-threonine was converted to l-isoleucine, which was secreted up to 30 g/l into the culture medium.