Biology subjects

Salvachua, D.

Publications and source records attributed to Salvachua, D..

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↗

bioRxiv2026

Pathway selection for arabinose utilization in Pseudomonas putida reveals a rate-yield tradeoff in muconic acid production from lignocellulosic sugars

Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.

synthetic biology↗

bioRxiv2025

The redefined role of PhaG and CoA ligases in medium-chain-length 3-hydroxy acid and polyhydroxyalkanoate production in Pseudomonas putida

Under nutrient starvation conditions, many microorganisms such as Pseudomonas putida store excess carbon in medium-chain-length polyhydroxyalkanoate (mcl-PHA) granules. Biosynthesis of PHAs from the fatty acid biosynthetic pathway requires PhaG, which has long been thought to encode a 3-hydroxyacyl-ACP:CoA transferase, and PhaC, the PHA polymerase. Although this pathway has been extensively studied, the exact role of PhaG remains inconclusive. In this work we present in vitro biochemical and in vivo genetic evidence demonstrating that PhaG functions as a 3-hydroxyacyl-ACP thiolase, producing 3-hydroxyacids rather than 3-hydroxyacyl-CoA. We identified two CoA ligases, fadD1 and alkK, essential for conversion of mcl-3-hydroxyacids to 3-hydroxyacyl-CoA, and thus PHA production. Taken together, this redefines the PHA biosynthetic pathway to include PhaG-dependent hydrolysis of hydroxyacyl-ACP, ligation of the 3-hydroxyacid to coenzyme-A, and polymerization by PhaC. Using these insights, we engineered P. putida to produce 3.7 g/L extracellular 3-hydroxyacids, which can be used for chemical synthesis of performance-advantaged polymers.

microbiology↗