(532d) Bioconversion of Lignin Streams into Muconate By Engineered Pseudomonas Putida

Lignin is a carbon-rich stream from biorefineries that is underutilized because of its recalcitrance. Isolated and depolymerized lignin contains many oligomeric and monomeric compounds that can serve as substrates for downstream processes. To evaluate the use of microbial species as biocatalysts that convert these compounds into commodity chemicals of higher value, we engineered a strain of Pseudomonas putida to convert 4-hydroxybenzoate (4HB) into muconate. Muconate has been identified by the US Department of Energy as a key building block chemical and is a precursor to a variety of products such as nylon and polyethylene terephthalate (PET).

We measured uptake and secretion rates of three strains: 1) wild type KT2440, 2) a strain engineered with a heterologous pathway enabling the bioconversion of 4HB to muconate, and 3) a derivative of the muconate pathway strain that removes competing pathways routing carbons from 4HB into biomass. We performed stable isotope carbon labeling experiments for these three strains in glucose media with and without 4HB and obtained mass distributions measurements for as many as 31 metabolites, including amino acids and metabolism intermediates. Using steady-state13C Metabolic Flux Analysis (13C-MFA) for a medium-size atom mapping network, we elucidated the fluxes for each strain with and without 4HB which revealed differences in the TCA cycle. Differential proteomics further highlighted dissimilarities of these strains under the two growth conditions. We evaluated bottlenecks that occur in the aromatic catabolic pathway, examined catabolism and toxicity tolerance, and here discuss the overall suitability of P. putida for catabolism of lignin-rich streams.