RB-TnSeq Identifies Genetic Targets for Improved Tolerance of seudomonas putida Towards Compounds Relevant to Lignin Conversion

Andrew Borchert, Alissa Bleem, Gregg Beckham

Research output: Contribution to journalArticlepeer-review

4 Scopus Citations


Lignin-derived mixtures intended for bioconversion commonly contain high concentrations of aromatic acids, aliphatic acids, and salts. The inherent toxicity of these chemicals places a significant bottleneck upon the effective use of microbial systems for the valorization of these mixtures. Pseudomonas putida KT2440 can tolerate stressful quantities of several lignin-related compounds, making this bacterium a promising host for converting these chemicals to valuable bioproducts. Nonetheless, further increasing P. putida tolerance to chemicals in lignin-rich substrates has the potential to improve bioprocess performance. Accordingly, we employed random barcoded transposon insertion sequencing (RB-TnSeq) to reveal genetic determinants in P. putida KT2440 that influence stress outcomes during exposure to representative constituents found in lignin-rich process streams. The fitness information obtained from the RB-TnSeq experiments informed engineering of strains via deletion or constitutive expression of several genes. Namely, ΔgacAS, ΔfleQ, ΔlapAB, ΔttgR::Ptac:ttgABC, Ptac:PP_1150:PP_1152, ΔrelA, and ΔPP_1430 mutants showed growth improvement in the presence of single compounds, and some also exhibited greater tolerance when grown using a complex chemical mixture representative of a lignin-rich chemical stream. Overall, this work demonstrates the successful implementation of a genome-scale screening tool for the identification of genes influencing stress tolerance against notable compounds within lignin-enriched chemical streams, and the genetic targets identified herein offer promising engineering targets for improving feedstock tolerance in lignin valorization strains of P. putida KT2440.

Original languageAmerican English
Pages (from-to)208-218
Number of pages11
JournalMetabolic Engineering
StatePublished - May 2023

Bibliographical note

Publisher Copyright:
© 2023 International Metabolic Engineering Society

NREL Publication Number

  • NREL/JA-2A00-86010


  • Acid tolerance
  • Biological funneling
  • Lignin valorization
  • Pseudomonas putida KT2440
  • Stress tolerance


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