Bioproduction Research Institute, National Institute of Advanced Industrial Sciences and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.
J Biosci Bioeng. 2012 Jul;114(1):38-44. doi: 10.1016/j.jbiosc.2012.02.029. Epub 2012 May 5.
Sugar derived from biomass is usually a mixture of glucose and other sugars. When mixed sugars are fed to Escherichia coli, glucose is preferentially utilized while other sugars remain unutilized. This phenomenon is known as carbon catabolite repression (CCR). To utilize mixed sugars effectively, we isolated a new E. coli mutant that is negative for CCR. The mutant strain was revealed to have a nucleotide substitution at the promoter region of mlc encoding a global transcriptional repressor for carbohydrate metabolism. The identified mutation, named mlc∗, was a promoter-up type, and the mlc∗ promoter exhibited 17-fold higher activity than the wild-type mlc promoter. Therefore, the mlc∗ mutation causes Mlc overexpression and a shortage of PtsG, which is a glucose-specific permease that is repressed by Mlc. The disruption of ptsG (ΔptsG) is known to induce a CCR-negative phenotype; the mlc∗ strain also exhibits the same phenotype via the same mechanism. As a sample application of the mlc∗ strain, we produced isobutanol from mixed sugars. Using glucose-xylose mixed sugar, the mlc∗ strain produced 1.4-fold more isobutanol than the parental wild-type strain. Also, the mlc∗ strain produced similar or greater amounts of isobutanol than other CCR-negative strains, ΔptsG and crp∗ (crp∗, encoding the constitutive-active mutant of cAMP receptor protein). In conclusion, the mlc∗ strain is a new CCR-negative strain that is useful for producing valuable compounds from mixed sugars.
生物质衍生的糖通常是葡萄糖和其他糖的混合物。当混合糖被喂食给大肠杆菌时，葡萄糖优先被利用，而其他糖则未被利用。这种现象被称为碳分解代谢物阻遏（CCR）。为了有效利用混合糖，我们分离出一种新的大肠杆菌突变体，该突变体对 CCR 呈阴性。该突变菌株在编码碳水化合物代谢全局转录抑制剂的 mlc 基因的启动子区域发生了核苷酸取代。鉴定出的突变，命名为 mlc∗，是一种启动子向上型，mlc∗启动子的活性比野生型 mlc 启动子高 17 倍。因此，mlc∗突变导致 Mlc 过表达和 PtsG 短缺，PtsG 是一种受 Mlc 抑制的葡萄糖特异性通透酶。ptsG 的缺失（ΔptsG）已知会诱导 CCR 阴性表型；mlc∗菌株也通过相同的机制表现出相同的表型。作为 mlc∗菌株的一个应用实例，我们从混合糖中生产异丁醇。使用葡萄糖-木糖混合糖，mlc∗菌株比亲本野生型菌株生产的异丁醇多 1.4 倍。此外，mlc∗菌株产生的异丁醇量与其他 CCR 阴性菌株（ΔptsG 和 crp∗）相似或更多，crp∗编码 cAMP 受体蛋白的组成性激活突变体。总之，mlc∗菌株是一种新的 CCR 阴性菌株，可用于从混合糖中生产有价值的化合物。
