Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Poly (ethylene terephthalate) (PET) is a widely used plastic that can be broken down into terephthalic acid (TPA) and ethylene glycol (EG).
• These monomers can be recycled into new PET (closed-loop) or converted into valuable products (open-loop).
• Pseudomonas putida KT2440 is a bacterium being engineered for PET monomer conversion.

Purpose of the Study:

• To improve the ability of Pseudomonas putida KT2440 to metabolize terephthalic acid (TPA) using adaptive laboratory evolution (ALE).
• To identify genetic modifications that enhance bacterial growth and TPA utilization for improved PET upcycling.

Main Methods:

• An automation-enabled ALE campaign was conducted on P. putida strains using TPA, a TPA and EG mixture, and glucose.
• Evolved strains were analyzed using whole-genome re-sequencing to identify genetic mutations.
• Key mutations were reverse-engineered and tested for their impact on TPA utilization.

Main Results:

• ALE significantly increased growth rates on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, over ~350 generations.
• The best performing evolved isolates showed growth rate increases of 0.15-0.20 h⁻¹ on TPA and TPA-EG.
• Whole-genome sequencing revealed converged mutations in global regulators (gacS, gacA, turA) and rearrangements affecting heterologous PET catabolic genes (tphABII).

Conclusions:

• ALE is effective in enhancing TPA catabolism in P. putida.
• Five genetic interventions, including deletions of global regulators and duplication of pathway genes, were identified to improve TPA utilization.
• These findings contribute to developing robust whole-cell biocatalysts for PET upcycling.