Analysis and Manipulation of Aspartate Pathway Genes for l -Lysine Overproduction from Methanol by Bacillus methanolicus

We investigated the regulation and roles of six aspartate pathway genes inl -lysine overproduction inBacillus methanolicus :dapG , encoding aspartokinase I (AKI);lysC , encoding AKII;yclM , encoding AKIII;asd , encoding aspartate semialdehyde dehydrogenase;dapA , encoding dihydrodipicolinate synthase; andlysA , encodingmeso -diaminopimelate decarboxylase. Analysis of the wild-type strain revealed thatin vivo lysC transcription was repressed 5-fold byl -lysine and induced 2-fold bydl -methionine added to the growth medium. Surprisingly,yclM transcription was repressed 5-fold bydl -methionine, while thedapG ,asd ,dapA , andlysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that thel -lysine-overproducing classicalB. methanolicus mutant NOA2#13A52-8A66 has—in addition to ahom-1 mutation—chromosomal mutations in thedapG coding region and in thelysA promoter region. No mutations were found in itsdapA ,lysC ,asd , andyclM genes. The mutantdapG gene product had abolished feedback inhibition bymeso -diaminopimelatein vitro , and thelysA mutation was accompanied by an elevated (6-fold)lysA transcription levelin vivo . Moreover,yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important forl -lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increasedl -lysine production levels.