Overproduction of L-Lysine from methanol by Methylobacillus glycogenes derivatives carrying a plasmid with a mutated dapA gene.
The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.
Motoyama
H
H
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Asahi-machi, Machida-shi, Tokyo 194, Japan. hmotoyama@kyowa.co.jp
Yano
H
H
Terasaki
Y
Y
Anazawa
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism*; D020631:Methylobacillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine; D000432:Methanol