SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, 7465 Trondheim, Norway.
Appl Environ Microbiol. 2011 Sep;77(17):6020-6. doi: 10.1128/AEM.05093-11. Epub 2011 Jul 1.
We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.
我们研究了天冬氨酸途径基因在巴氏甲烷八叠球菌 L-赖氨酸过量生产中的调控和作用：编码天冬氨酸激酶 I（AKI）的 dapG；编码 AKII 的 lysC；编码 AKIII 的 yclM；编码天冬氨酸半醛脱氢酶的 asd；编码二氢二吡啶羧酸合酶的 dapA；以及编码 meso-二氨基庚二酸脱羧酶的 lysA。对野生型菌株的分析表明，lysC 转录在体内被 L-赖氨酸抑制 5 倍，并被添加到生长培养基中的 dl-蛋氨酸诱导 2 倍。令人惊讶的是，yclM 转录被 dl-蛋氨酸抑制 5 倍，而 dapG、asd、dapA 和 lysA 基因不受任何天冬氨酸途径氨基酸的显著抑制。我们表明，除了 hom-1 突变外，L-赖氨酸高产经典巴氏甲烷八叠球菌突变体 NOA2#13A52-8A66 还在 dapG 编码区和 lysA 启动子区发生了染色体突变。其 dapA、lysC、asd 和 yclM 基因未发现突变。突变 dapG 基因产物在体外失去了 meso-二氨基庚二酸的反馈抑制，而 lysA 突变伴随着体内 lysA 转录水平的升高（6 倍）。此外，突变株 NOA2#13A52-8A66 中 yclM 的转录水平比野生型菌株增加了 16 倍。野生型和突变型天冬氨酸途径基因的过表达表明，在摇瓶试验中，所有 6 个基因对 L-赖氨酸的过量生产都很重要，并且效果取决于所测试的遗传背景。多达三个基因的偶联过表达导致（超过 80 倍）增加的 L-赖氨酸生产水平。
