RB-TnSeq uncovers genetic targets for engineering improved tolerance against notable lignin-rich chemical stream constituents in Pseudomonas putida

Lignin-enriched biomass hydrolysates 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 biological systems for the valorization of these mixtures. The ability for Pseudomonas putida KT2440 to tolerate stressful quantities of several lignin-derived chemicals has led to increasing interest in using P. putida as a biological chassis for the conversion of sugars and aromatic acids into valuable commodities. Nonetheless, increasing P. putida tolerance to chemicals within lignin-enriched biomass hydrolysates has the potential to improve biocatalytic outcomes. Accordingly, this work employed random barcoded transposon insertion sequencing (RB-TnSeq) to uncover Pseudomonas putida KT2440 genetic determinants influencing stress outcomes during exposure to several constituents found in lignin-enriched process streams as well as for frequent valorization products created from many of these chemical constituents. Stress outcomes was tested for 4-coumarate, ferulate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, vanillin, vanillate, NaCl, Na2O4, acetate, lactate, glycolate, cis,cis-muconate, protecatechuate, beta-ketoadipate, and levulinate.