RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion

Borchert, Andrew J.; Bleem, Alissa; Beckham, Gregg T.

doi:10.1016/j.ymben.2023.04.007

RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion

Journal Article · Thu Apr 13 00:00:00 EDT 2023 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2023.04.007· OSTI ID:1974027

Borchert, Andrew J. ^[1]; Bleem, Alissa ^[1]; ^[1]

National Renewable Energy Lab. (NREL), Golden, CO (United States). Renewable Resources and Enabling Science Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation (CBI)

We report 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::P_tac:ttgABC, Ptac:PP_1150:PP_1152, ..delta..relA, 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.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO)

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1974027

Report Number(s):: NREL/JA-2A00-86010; MainId:86783; UUID:a6cae123-7a68-4a92-8601-59739b39f930; MainAdminID:69520

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 77; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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