Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-37 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
J Biosci Bioeng. 2012 Jan;113(1):63-9. doi: 10.1016/j.jbiosc.2011.09.014. Epub 2011 Oct 20.
Although the facultative chemolithoautotrophic Cupriavidus necator (formerly Ralstonia eutropha) wild strain H16 is potentially useful as a host for metabolic engineering aimed at polyhydroxyalkanoate production, this organism is deficient in assimilating glucose, a major sugar in non-edible cellulosic resources. Growth properties of C. necator H16 harboring heterologous glf (encoding glucose-facilitated diffusion transporter) and glk (encoding glucokinase) from Zymomonas mobilis strongly suggested that the lack of glucose-utilization ability of C. necator H16 was caused by deficiency of both glucose-uptake and phosphorylation abilities. Next examination focused on previously unknown mutation points in a glucose-utilizing mutant of C. necator NCIMB 11599. Direct sequencing of a region of genes for putative N-acetylglucosamine-specific phosphoenolpyruvate-dependent phosphotransferase system and its upstream region identified a missense mutation in nagE corresponding to Gly265Arg in the EIIC-EIIB component, and a nonsense mutation in nagR encoding a putative GntR-type transcriptional regulator. Further analyses demonstrated that the glucose-utilization ability of C. necator NCIMB 11599 is attributed to extended sugar specificity of the mutated NagE and derepression of nagFE expression by inactivation of NagR. The mutation in nagE and disruption of nagR were then introduced onto chromosome 1 of wild strain H16 by homologous recombination. The resulting engineered strain C. necator nagE_G265R∆nagR exhibited comparable growth and poly(3-hydroxybutyrate) accumulation on glucose to those of the wild strain on fructose, demonstrating successful reconstitution of functional glucose-uptake and phosphorylation system. This recombinant strain is expected to be useful in further engineering for efficient production of PHAs from inexpensive biomass resources.
尽管兼性化能自养的恶臭假单胞菌(以前称为罗尔斯通氏菌)野生株 H16 具有作为代谢工程宿主的潜力,旨在生产聚羟基烷酸,但该生物体在同化葡萄糖方面存在缺陷,葡萄糖是不可食用纤维素资源中的主要糖。携带来自运动发酵单胞菌的异源 glf(编码葡萄糖促进扩散转运蛋白)和 glk(编码葡萄糖激酶)的 C. necator H16 的生长特性强烈表明,C. necator H16 缺乏葡萄糖利用能力是由于葡萄糖摄取和磷酸化能力均不足所致。接下来的检查集中在恶臭假单胞菌 NCIMB 11599 的一个葡萄糖利用突变体的未知突变点上。对假定的 N-乙酰葡萄糖胺特异性磷酸烯醇丙酮酸依赖性磷酸转移酶系统及其上游区域的基因区域进行直接测序,确定了 EIIC-EIIB 组件中 nagE 对应于 Gly265Arg 的错义突变,以及编码假定 GntR 型转录调节剂的 nagR 中的无义突变。进一步的分析表明,恶臭假单胞菌 NCIMB 11599 的葡萄糖利用能力归因于突变的 NagE 的扩展糖特异性以及 NagR 失活导致 nagFE 表达的去阻遏。然后通过同源重组将 nagE 中的突变和 nagR 的缺失引入到野生株 H16 的染色体 1 上。由此产生的工程菌株 C. necator nagE_G265R∆nagR 在葡萄糖上表现出与野生株在果糖上相当的生长和聚(3-羟基丁酸酯)积累,表明功能葡萄糖摄取和磷酸化系统的成功重建。该重组菌株有望在进一步的工程设计中用于从廉价生物质资源高效生产 PHAs。