Williams-Rhaesa Amanda M, Awuku Nanaakua K, Lipscomb Gina L, Poole Farris L, Rubinstein Gabriel M, Conway Jonathan M, Kelly Robert M, Adams Michael W W
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Extremophiles. 2018 Jul;22(4):629-638. doi: 10.1007/s00792-018-1023-x. Epub 2018 May 24.
Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.
对同源和异源基因表达进行调控对于目标微生物的精确基因操作和代谢工程至关重要。然而，在处理非模式微生物时，通常没有可诱导启动子可供选择。这些微生物包括极端嗜热的纤维素分解细菌，它们对于将可再生木质纤维素转化为生物燃料和化学品具有重要意义。事实上，一旦实现转化，这些生物体遗传系统的改进往往就会停止。本研究扩展了可用于对嗜热栖热放线菌进行基因工程改造的工具，嗜热栖热放线菌是已知的最嗜热的纤维素降解菌，能在未经预处理的植物生物质上生长至90°C。利用一个天然的木糖诱导型（P ）启动子来控制编码乳酸脱氢酶的报告基因（ldh）的表达。与以葡萄糖为底物时相比，在木糖存在的情况下，P -ldh构建体导致ldh表达增加（高20倍）以及乳酸脱氢酶活性增加（高32倍）。最后，重组嗜热栖热放线菌菌株生长过程中的乳酸产量与初始木糖浓度成正比，这表明使用该木糖诱导系统现在可以实现基因的可调表达。这项研究代表了将嗜热栖热放线菌用作利用可再生生物质进行生物技术应用的潜在平台微生物的一个重要进展。
