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

Werner, Allison Z.; Avina, Young-Saeng C.; Johnsen, Josefin; Bratti, Felicia; Alt, Hannah M.; Mohamed, Elsayed T.; Clare, Rita; Mand, Thomas D.; Guss, Adam M.; Feist, Adam M.; Beckham, Gregg T.

doi:10.1016/j.ymben.2024.12.006

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

Journal Article · Tue Dec 17 00:00:00 EST 2024 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2024.12.006· OSTI ID:2496615

Werner, Allison Z. ^[1]; Avina, Young-Saeng C. ^[2]; Johnsen, Josefin ^[3]; Bratti, Felicia ^[1]; Alt, Hannah M. ^[4]; Mohamed, Elsayed T. ^[3]; Clare, Rita ^[1]; Mand, Thomas D. ^[5]; Guss, Adam M. ^[5]; Feist, Adam M. ^[6]; ^[1]

National Renewable Energy Laboratory (NREL), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); Technical Univ. of Denmark, Lyngby (Denmark)
Technical Univ. of Denmark, Lyngby (Denmark)
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
BOTTLE Consortium, Golden, CO (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Technical Univ. of Denmark, Lyngby (Denmark); Univ. of California, San Diego, CA (United States)

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h^-1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB_II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB_II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

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This content will become publicly available on Wed Dec 17 00:00:00 EST 2025

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Energy Efficiency Office. Advanced Materials & Manufacturing Technologies Office (AMMTO); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO); Novo Nordisk Foundation (NNF)

Grant/Contract Number:: AC36-08GO28308; AC05-00OR22725; NNF20CC0035580

OSTI ID:: 2496615

Report Number(s):: NREL/JA-2A00-91753; MainId:93531; UUID:9fe1d796-b040-4911-a76d-cbdbdca4303e; MainAdminId:75571

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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