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Jakobsen, OM, Brautaset, T, Degnes, KF, Heggeset, TM, Balzer, S, Flickinger, MC, Valla, S and Ellingsen, TE (2009) Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus. Appl. Environ. Microbiol. 75:652-61
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 micromol/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.
Animals; Aspartate Kinase/genetics; Aspartate Kinase/isolation & purification; Aspartate Kinase/metabolism; Aspartate-Semialdehyde Dehydrogenase/genetics; Aspartate-Semialdehyde Dehydrogenase/metabolism; Aspartic Acid/metabolism; Bacillus/enzymology; Bacillus/genetics; Bacillus/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; Diaminopimelic Acid/pharmacology; Enzyme Inhibitors/pharmacology; Gene Dosage; Gene Expression; Hydro-Lyases/genetics; Hydro-Lyases/metabolism; Inhibitory Concentration 50; Kinetics; Lysine/biosynthesis; Methionine/biosynthesis; Molecular Sequence Data; Sequence Analysis, DNA; Threonine/pharmacology