Combinatorial pathway engineering using multiplex Serine recombinase-Assisted Genome Engineering (mSAGE)

Low efficiency of DNA integration into the chromosome limits multiplexed bacterial genetic engineering. We address this by establishing multiplexed Serine recombinase-Assisted Genome Engineering (mSAGE), enabling simultaneous, site-specific insertion of three DNAs into the chromosome of Pseudomonas putida KT2440. We construct a combinatorial library of isophthalate catabolism and transport genes through a single, pooled transformation of three gene libraries, rapidly identifying the best combination of genes for biodegradation of this plastic comonomer.