Overexpression of Wild-Type Aspartokinase Increases L-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids L-methionine, L-threonine, L-isoleucine, and L-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased L-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of L-methionine (less than 0.5 g/liter) and L-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 mu mol/min/mg protein) and Km values for L-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [ IC(50)], 0.1 mM) and by L-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by L-threonine (IC(50), 4 mM) and by L-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on L-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving L-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing L-lysine production by overexpression of a wild-type AK.