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

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.