(191bb) Engineering Metabolic Pathways By Using Standardized DNA Parts

Almost all metabolic engineering practices require plasmid construction. Currently, tens of thousands of research laboratories across the globe are constructing plasmids every day in an inefficient way – researchers customize materials they need, pay commercial companies to synthesize them from scratch (oligos and/or genes), wait for the materials to be delivered, and assemble the materials in their own laboratories.

A more efficient way of constructing plasmids is through assembly of standardized DNA parts, which are from a user-defined library and readily available. Here, we developed a scarless DNA assembly method that can assemble up to 12 standardized DNA parts in any order. Our method does not need to avoid any forbidden restriction enzyme site, and is compatible with multiple-tier DNA assembly, which makes it to be advantageous to all existing methods that use standardized DNA parts (e.g. BioBricks assembly and BASIC). To demonstrate the usefulness of this method, we built a library of DNA parts for metabolic engineering applications, and used them to overproduce isoprenoid and aromatic compounds in commonly used species (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae). In this era of CRISPR-Cas9, we also designed a large number of parts for this genome editing method, and used them to construct some plasmid-free microbes that have more stable fermentation characteristics in metabolic engineering applications. Through sharing and improving this library of DNA parts, our DNA assembly method would make it cheaper and faster to engineer microbial metabolic pathways for overproduction of valuable chemicals.