title: Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.
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 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.
authors.affiliation: Department of Biotechnology, SINTEF Materials and Chemistry, Trondheim, Norway. oyvind.m.jakobsen@sintef.no
authors.forename: Oyvind M
authors.initials: OM
authors.lastname: Jakobsen
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
authors.forename: Kristin F
authors.initials: KF
authors.lastname: Degnes
authors.forename: Tonje M B
authors.initials: TM
authors.lastname: Heggeset
authors.forename: Simone
authors.initials: S
authors.lastname: Balzer
authors.forename: Michael C
authors.initials: MC
authors.lastname: Flickinger
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
authors.forename: Trond E
authors.initials: TE
authors.lastname: Ellingsen
mesh_terms: D000818:Animals; D001222:Aspartate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003960:Diaminopimelic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018628:Gene Dosage; D015870:Gene Expression; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008239:Lysine / Q000096:biosynthesis*; D008715:Methionine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013912:Threonine / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D013912:Threonine; D001224:Aspartic Acid; D003960:Diaminopimelic Acid; D008715:Methionine; D001223:Aspartate-Semialdehyde Dehydrogenase; D001222:Aspartate Kinase; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D008239:Lysine