Englaender Jacob A, Jones J Andrew, Cress Brady F, Kuhlman Thomas E, Linhardt Robert J, Koffas Mattheos A G
Department of Biological Sciences, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
ACS Synth Biol. 2017 Apr 21;6(4):710-720. doi: 10.1021/acssynbio.6b00350. Epub 2017 Jan 13.
Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.
染色体整合为外源蛋白和代谢途径的表达提供了一种无需选择的替代DNA质粒的方法。游离质粒DNA使用方便，但存在缺点，包括代谢负担增加以及需要抗生素形式的选择。长期以来，大肠杆菌一直用于外源蛋白的表达以及通过完整代谢途径的表达来生产有价值的代谢物。将编码荧光报告蛋白mCherry的基因整合到大肠杆菌染色体上的四个基因组位点，以测量每个位点的蛋白表达。与在高拷贝质粒上表达的基因相比，表达水平范围为25%至500%。DNA的模块化表达是通过代谢工程优化代谢物生产最常用的方法之一。通过结合最近开发的将大型合成DNA构建体整合到基因组中的方法，我们能够将两条外源途径整合到相同的四个基因组位点。我们已经证明，只有一个基因组位点导致了紫菌素的产生，并且所有四个位点都通过TAL途径产生了反式肉桂酸。
