Methods and applications for assembling large DNA constructs

The construction of large DNA molecules that encode pathways, biological machinery, and entire genomes has been limited to the reproduction of natural sequences. However, now that robust methods for assembling hundreds of DNA fragments into constructs >20 kb are readily available, optimization of large genetic elements for metabolic engineering purposes is becoming more routine. Here, various DNA assembly methodologies are reviewed and some of their potential applications are discussed. We tested the potential of DNA assembly to install rational changes in complex biosynthetic pathways, their potential for generating complex libraries, and consider how various strategies are applicable to metabolic engineering.