Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
Appl Environ Microbiol. 2013 Feb;79(3):774-82. doi: 10.1128/AEM.02578-12. Epub 2012 Nov 16.
Despite numerous approaches for the development of l-threonine-producing strains, strain development is still hampered by the intrinsic inefficiency of metabolic reactions caused by simple diffusion and random collisions of enzymes and metabolites. A scaffold system, which can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, was reported to be one of the most promising solutions. Here, we report an improvement in l-threonine production in Escherichia coli using a DNA scaffold system, in which a zinc finger protein serves as an adapter for the site-specific binding of each enzyme involved in l-threonine production to a precisely ordered location on a DNA double helix to increase the proximity of enzymes and the local concentration of metabolites to maximize production. The optimized DNA scaffold system for l-threonine production significantly increased the efficiency of the threonine biosynthetic pathway in E. coli, substantially reducing the production time for l-threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the host strain by reducing the intracellular concentration of toxic intermediates, such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial metabolic pathways as well as for the production of many useful biomaterials.
尽管已经提出了许多用于生产 L-苏氨酸的菌株的方法，但由于酶和代谢物的简单扩散和随机碰撞导致的代谢反应固有低效性，菌株的开发仍然受到阻碍。支架系统可以促进代谢酶的接近，并增加中间产物的局部浓度，被认为是最有前途的解决方案之一。在这里，我们报告了使用 DNA 支架系统提高大肠杆菌中 L-苏氨酸生产的改进，其中锌指蛋白作为接头，用于将参与 L-苏氨酸生产的每种酶特异性结合到 DNA 双螺旋上的精确有序位置，以增加酶的接近度和代谢物的局部浓度，从而最大化生产效率。用于 L-苏氨酸生产的优化 DNA 支架系统显著提高了大肠杆菌中苏氨酸生物合成途径的效率，大大缩短了 L-苏氨酸的生产时间（超过 50%）。此外，该 DNA 支架系统通过降低有毒中间产物（如高丝氨酸）的细胞内浓度来提高宿主菌株的生长速度。我们的 DNA 支架系统可作为构建和优化人工代谢途径以及生产许多有用生物材料的平台技术。
