Genomic Analysis for the Mechanism of Leucine Overproduction in a Corynebacterium  Glutamicum

Amino acid industry has a long history for over 50 years. At present, many kinds of amino acid are produced by fermentation. In the early days of amino acid production, the producer strains were bred by repeated conventional mutagenesis and selection. However, due to the limited technology available at the time, little was studied for the genetic analysis of the strain to reveal the mechanism of overproduction.

The wild type strain has a regulation system to prevent the overproduction of each amino acid; feedback inhibition and repression at each key-enzyme step in the bio-synthetic pathway. Therefore, to achieve the overproduction, mutation in the genes of coding the key enzyme to release of the feedback inhibition and repression are essential. The most important key-enzymes for leucine production are α-isopropyl malate synthase (IPMS) and acetohydroxylate synthase (AHAS), which are coded by leuA and ilvBN genes, respectively.

Corynebacterium glutamicum strain NB-12 is a leucine producer that is bred by conventional mutagenesis. Whole genome sequence was determined by SOLiD system and compared with the wild type strain. A single mutation was observed in the coding regions of both the leuA (G564D) and ilvBN (G235D) genes. These findings suggest that the mutations lead to the release of feedback inhibition: in fact, the introduction of the two mutant genes into wild type E.coli is resulted in leucine accumulation. However, concerning feedback repression, no mutation was observed in the promoter region of either gene. Instead, NB-12 is known to be an Ile auxotroph, and a mutation was observed in the ilvA gene encoding threonine dehydratase (A95T). These results suggest that the addition of limited amount of isoleucine leads to the release of feedback repression of the genes encoding these key enzymes. In the present study, specific mutations and mechanism of leucine overproduction will be discussed.