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

Abstract

ABSTRACT We investigated the regulation and roles of six aspartate pathway genes in l -lysine overproduction in Bacillus methanolicus : dapG , encoding aspartokinase I (AKI); lysC , encoding AKII; yclM , encoding AKIII; asd , encoding aspartate semialdehyde dehydrogenase; dapA , encoding dihydrodipicolinate synthase; and lysA , encoding meso -diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l -lysine and induced 2-fold by dl -methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl -methionine, while the dapG , asd , dapA , and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l -lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA , lysC , asd , and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso -diaminopimelate in vitro , and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in 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 for l -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) increased l -lysine production levels.