Jakobsen Oyvind M, Brautaset Trygve, Degnes Kristin F, Heggeset Tonje M B, Balzer Simone, Flickinger Michael C, Valla Svein, Ellingsen Trond E
Department of Biotechnology, SINTEF Materials and Chemistry, Trondheim, Norway.
Appl Environ Microbiol. 2009 Feb;75(3):652-61. doi: 10.1128/AEM.01176-08. Epub 2008 Dec 5.
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.
天冬氨酸激酶（AK）控制着碳流向天冬氨酸途径，用于生物合成氨基酸L-甲硫氨酸、L-苏氨酸、L-异亮氨酸和L-赖氨酸。我们在此报告了甲醇芽孢杆菌MGA3中天冬氨酸途径的四个基因（asd，编码天冬氨酸半醛脱氢酶；dapA，编码二氢二吡啶甲酸合酶；dapG，编码AKI；yclM，编码AKIII）的克隆。与已知的AKII基因lysC一起，dapG和yclM在该生物体中形成了一组三个AK基因。dapG、lysC和yclM的过表达分别使野生型甲醇芽孢杆菌菌株MGA3中的L-赖氨酸产量提高了2倍、10倍和60倍（分别对应于11克/升），且对比生长速率没有负面影响。在所有重组菌株中，L-甲硫氨酸（低于0.5克/升）和L-苏氨酸（低于0.1克/升）的产量都很低。对AK蛋白进行了纯化，生化分析表明它们具有相似的V(max)值（在47至58微摩尔/分钟/毫克蛋白之间）和对L-天冬氨酸的K(m)值（在1.9至5.0毫摩尔之间）。AKI和AKII分别被内消旋二氨基庚二酸（50%抑制浓度[IC(50)]，0.1毫摩尔）和L-赖氨酸（IC(50)，0.3毫摩尔）变构抑制。AKIII被L-苏氨酸（IC(50)，4毫摩尔）和L-赖氨酸（IC(50)，5毫摩尔）抑制，并且在低浓度下这两种氨基酸同时存在时，该酶会受到协同抑制。讨论了对体内L-赖氨酸产量的影响与各个AK蛋白体外生化特性之间的相关性。这是通过甲醇芽孢杆菌的代谢工程提高L-赖氨酸产量的首个实例，也是通过野生型AK的过表达大幅提高L-赖氨酸产量的首次记录。
