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DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

Shao, Zengyi, Zhao, Hua, Zhao, Huimin
Nucleic acids research 2009 v.37 no.2 pp. e16
Saccharomyces cerevisiae, biochemical pathways, biosynthesis, chromosomes, genes, genomics, homologous recombination, metabolic engineering, methodology, plasmids, recombinant DNA, synthetic biology, xylose, yeasts, zeaxanthin
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 (~9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (~11 kb DNA consisting of five genes) and a functional combined D-xylose utilization and zeaxanthin biosynthesis pathway (~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.