Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

TLDR

Synthetic biology requires efficient methods to assemble and modify DNA parts into functional devices. The study demonstrates a rapid ϕC31 integrase-based method for assembling DNA fragments and shows its use for building, optimizing, and modifying metabolic pathways. The approach employs six orthogonal attP/attB recombination sites with distinct overlaps to assemble up to five fragments in a defined order in a single reaction. The integrase-based assembly is highly efficient, generating large combinatorial libraries and cassette arrays that can be further recombined.

Abstract

Abstract Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.