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

Huenemann, J. D.; Carroll, A. L.; Elmore, J. R.; Dexter, G.; Mand, T.; Klingeman, D. M.; Alexander, W.; Guss, A. M.

2025-07-11 microbiology
10.1101/2025.07.11.664204 bioRxiv
Show abstract

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.

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