Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.
Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.
Ogawa-Miyata
Y
Y
Fermentation & Biotechnology Laboratories, Ajinomoto Co. Inc., Kawasaki-shi, Japan.
Kojima
H
H
Sano
K
K
D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005246:Feedback; D006358:Hot Temperature; D008969:Molecular Sequence Data; D013912:Threonine / Q000096:biosynthesis*
D017931:DNA Primers; D013912:Threonine; C116188:lysC aspartokinase; D001222:Aspartate Kinase