Biology subjects

Wilkes, R. A.

Publications and source records attributed to Wilkes, R. A..

3 recordsLinked to original sources

bioRxiv2026

Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

synthetic biology↗

bioRxiv2025

Quantitative Analysis of Coupled Carbon and Energy Metabolism for Lignin Carbon Utilization in Pseudomonas putida

Soil Pseudomonas species, which can thrive on lignin-derived phenolic compounds, are widely explored for biotechnology applications. Yet, there is limited understanding of how the native metabolism coordinates phenolic carbon processing with cofactor generation. Here, we achieve quantitative understanding of this metabolic balance through a multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate as a non-aromatic reference, proteomics data reveal >140-fold increase in proteins for transport and initial catabolism of each phenolic substrate, but metabolomics profiling reveals that bottleneck nodes in initial phenolic compound catabolism maintain more favorable cellular energy state. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling via pyruvate carboxylase promotes fluxes in the tricarboxylic acid cycle to provide 50-60% NADPH yield and 60-80% NADH yield, resulting in 2-fold higher ATP yield than for succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. The quantitative blueprint elucidated here explains deficient versus sufficient cofactor rebalancing during manipulations of key metabolic nodes in lignin valorization.

microbiology↗

bioRxiv2018

Metabolic Flux Hierarchy Prioritizes the Entner-Doudoroff Pathway for Carbohydrate Co-Utilization in Pseudomonas protegens Pf-5

The genetic characterization of Pseudomonas protegens Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here we employed 13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. Similar to other Pseudomonas species, P. protegens Pf-5 relied primarily on the Entner-Doudoroff (ED) pathway to connect initial glucose catabolism to downstream metabolic pathways. Flux quantitation determined that, in lieu of the direct phosphorylation of glucose by glucose kinase, phosphorylation of oxidized products of glucose (gluconate and 2-ketogluconate) towards the ED pathway accounted for over 90% of consumed glucose and greater than 35% of consumed glucose was secreted as gluconate and 2-ketogluconate. Consistent with the lack of annotated pathways for the initial catabolism of pentoses and galactose in P. protegens Pf-5, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of the hexoses was evident but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism toward fructose 6-phosphate, metabolite labeling patterns revealed that mannose-derived carbons specifically entered central carbon metabolism via fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were found to cycle backward through the upper Emden-Meyerhof-Parnas pathway to feed into the ED pathway. Therefore, the operational metabolic network for processing carbohydrates in P. protegens Pf-5 prioritizes flux through the ED pathway to channel carbons to downstream metabolic pathways.\n\nIMPORTANCESpecies of the Pseudomonas genus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here we investigated the metabolic network for carbohydrate metabolism in P. protegens Pf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through the network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the gene annotation of two different pathways in the genome. Elucidation of the constitutive metabolic network in P. protegens is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped Pseudomonas species.

microbiology↗