Shao Zengyi, Zhao Hua, Zhao Huimin
Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Nucleic Acids Res. 2009 Feb;37(2):e16. doi: 10.1093/nar/gkn991. Epub 2008 Dec 12.
The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional D-xylose utilization pathway (approximately 9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (approximately 11 kb DNA consisting of five genes) and a functional combined D-xylose utilization and zeaxanthin biosynthesis pathway (approximately 19 kb consisting of eight genes) with high efficiencies (70-100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.
编码完整生化途径或基因组的大型重组DNA的组装是一项重大挑战。在此，我们报告了一种新方法——DNA组装器，它能够通过酿酒酵母中的体内同源重组在单个步骤中组装完整的生化途径。我们表明，DNA组装器可以高效（70 - 100%）地在质粒或酵母染色体上快速组装功能性D - 木糖利用途径（由三个基因组成的约9 kb DNA）、功能性玉米黄质生物合成途径（由五个基因组成的约11 kb DNA）以及功能性D - 木糖利用和玉米黄质生物合成组合途径（由八个基因组成的约19 kb）。由于这种新方法仅需要简单的DNA制备和一步酵母转化，它在合成生物学、代谢工程和功能基因组学研究的生化途径构建中代表了一种强大的工具。
