Institut Pasteur, Département Génomes et Génétique, Paris, France.
Nucleic Acids Res. 2010 Aug;38(15):e153. doi: 10.1093/nar/gkq511. Epub 2010 Jun 9.
As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with 'regulatory' elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.
随着合成生物学领域的不断扩展，快速构建新生化途径的策略和工具将变得越来越有价值。由于当前知识水平的限制，复杂生物途径的纯理性设计具有固有局限性。从随机文库中选择最佳的遗传元件排列可能是成功工程的一种有效方法。在这里，我们提出了使用天然细菌位点特异性重组系统（整合子）的固有基因重排活性来构建和优化代谢途径。作为原理验证，我们在大肠杆菌中构建和优化了功能性色氨酸生物合成操纵子。trpA-E 基因与“调节”元件作为单个重组盒被递送到合成整合子平台中。整合酶介导的重组在一夜之间产生了成千上万的遗传组合。我们能够分离出大量显示不同适应性和色氨酸生产能力的排列。一些组合需要多达六次重组事件，并且在相同的背景下产生的色氨酸比天然基因顺序多 11 倍。
