Biology subjects

Ramirez, K. J.

Publications and source records attributed to Ramirez, K. J..

5 recordsLinked to original sources

bioRxiv2026

A pyoverdine-type metallophore is required for lanthanide-dependent growth in Pseudomonas putida

Lanthanides (Ln) are recently discovered cofactors for alcohol metabolism in a growing number of bacteria, including the soil-dwelling bacterium Pseudomonas putida, yet the mechanisms for Ln uptake are poorly defined. Here, we discovered that the non-ribosomal peptide synthase biosynthetic gene cluster, pvdLIJD, was essential to Ln-dependent growth in P. putida. Pyoverdine G4R and its precursor ferribactin were identified as pvdLIJD gene products with La-responsive abundances, and both chelated La as well as iron. Deletion of the putative pyoverdine outer membrane transport system encoded by fpvA and exbBD:tonB were strongly linked to Ln-dependent growth in a concentration-dependent manner. Global transcriptomes were remodeled in response to La, but weak evidence of transcriptional pvdLIJD regulation and no evidence of discernment between La provided as chloride versus oxide were observed. Overall, this work establishes pyoverdines as metallophores involved in La uptake, re-framing them from strictly iron-scavenging siderophores to dual-purpose metallophores with Ln-binding activity.

microbiology↗

bioRxiv2026

Expanding the catabolic capacity of Pseudomonas putida to acetovanillone, 5-carboxyvanillate, and vanillyl glyoxylate for muconate production from kraft lignin-derived aromatics

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C--O and C--C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 {+/-} 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 {+/-} 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 {+/-} 0.1 mol% through incorporation of three {beta}-5 cleavage products, in addition to traditional G-type monomers.

synthetic biology↗

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↗