Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis.
Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes. The leuA complementing fragment encoding the isopropylmalate synthase was sequenced. The leuA gene is 1,848 bp in size, encoding a polypeptide with an M(r) of 68,187. Upstream of leuA there is extensive hyphenated dyad symmetry and a putative leader peptide, which are features characteristic of attenuation control. In addition to leuA, the sequenced fragment contains an open reading frame with high coding probability whose disruption did not result in a detectable phenotype. Furthermore, the sequence revealed that this open reading frame separates leuA from lysC, which encodes the aspartate kinase initiating the synthesis of all amino acids of the aspartate family. The leuA gene was inactivated in three lysine-secreting strains by insertional mutagenesis. Fermentations were performed, and a roughly 50% higher lysine yield was obtained when appropriate leucine concentrations limiting for growth of the constructed strains were used.
Pátek
M
M
Institut für Biotechnologie 1, Jülich, Germany.
Krumbach
K
K
Eggeling
L
L
Sahm
H
H
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000378:metabolism; D007930:Leucine / Q000096:biosynthesis*; D008239:Lysine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D006836:Hydro-Lyases; C021455:3-isopropylmalate dehydratase; D007930:Leucine; D008239:Lysine