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

Abstract

ABSTRACT 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 μmol/min/mg protein) and K m 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.