Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.
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 C31 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. C31 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.
Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, University Avenue, Glasgow G12 8QQ, Scotland, UK, Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK, Department of Metabolic Biology, The John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK, Joint BioEnergy Institute, Emeryville, CA 94608, USA, Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Sean D
SD
Colloms
Christine A
CA
Merrick
Femi J
FJ
Olorunniji
W Marshall
WM
Stark
Margaret C M
MC
Smith
Anne
A
Osbourn
Jay D
JD
Keasling
Susan J
SJ
Rosser
D001435:Bacteriophages / Q000201:enzymology; D053898:Biosynthetic Pathways / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D023061:Gene Order; D019426:Integrases / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011995:Recombination, Genetic; D012270:Ribosomes / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods
D019426:Integrases