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Applied and Environmental Microbiology, February 2009, p. 652-661, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01176-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Øyvind M. Jakobsen,^1,2* Trygve Brautaset,¹ Kristin F. Degnes,¹ Tonje M. B. Heggeset,¹ Simone Balzer,^1,2 Michael C. Flickinger,^3, Svein Valla,² and Trond E. Ellingsen^1,2

Department of Biotechnology, SINTEF Materials and Chemistry, Trondheim, Norway,¹ Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway,² BioTechnology Institute, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota³

Received 27 May 2008/ Accepted 25 November 2008

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 [IC₅₀], 0.1 mM) and by L-lysine (IC₅₀, 0.3 mM), respectively. AKIII was inhibited by L-threonine (IC₅₀, 4 mM) and by L-lysine (IC₅₀, 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.

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* Corresponding author. Mailing address: Department of Biotechnology, SINTEF Materials and Chemistry, 7465 Trondheim, Norway. Phone: 47 932 33 047. Fax: 47 735 96 995. E-mail: oyvind.m.jakobsen{at}sintef.no

Published ahead of print on 5 December 2008.

Present address: Department of Microbiology, North Carolina State University, Raleigh, NC.

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Applied and Environmental Microbiology, February 2009, p. 652-661, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01176-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.