Pro proteomics, metabolomics, and 13C-fluxomics are applied to quantitatively compare central carbon and energy metabolism in wild-type P. putida and a muconate-producing strain for improved bioproduction from renewable feedstocks.
Abstract
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.
ABSTRACT Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon‐conserving alternative by cleaving fructose‐6‐phosphate and/or xylulose‐5‐phosphate into acetyl‐phosphate, which can subsequently be converted to acetyl‐coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO2 release. Here, we engineered this strategy in Bacillus methanolicus , a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B (pktB) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol‐to‐biomass yields by 18%–24% relative to controls and reduced biogenic CO2 production by 9%–12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid‐based expression. Together, these results show that PKT‐driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon‐conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.
Yael J. Toporek, J. Humphreys, Thomas J. Musselwhite et al.· Microbial Biotechnology· 0 citations
Lignin has potential as a sustainable feedstock to replace fossil fuels in chemical manufacturing. The coupling of chemical fractionation and biocatalysis has emerged as a promising technology to realize this potential. In this process, chemocatalytic fractionation of biomass or lignin yields heterogeneous mixtures of lignin-derived aromatic compounds (LDACs), which are subsequently funneled to target chemicals by microbial cell factories. The recent expansion of genetic toolkits for non-model bacteria offers burgeoning possibilities for engineering bespoke biocatalysts using natural LDAC degraders such as Rhodococcus aromaticivorans RHA1. Herein, we describe the development of an RHA1 biocatalyst to convert a softwood kraft lignin stream containing vanillin, vanillate and acetovanillone to muconic acid by leveraging Serine integrase-Assisted Genome Engineering (SAGE). We increased vanillin metabolism by co-expressing ligV, encoding a vanillin dehydrogenase, and RHA1's endogenous vanACB, encoding a vanillate O-demethylase, partially overcoming the vanillate bottleneck observed when expressing ligV alone. To funnel aromatics to muconic acid, we tested two aromatic acid decarboxylases, finding that AroY with EcdBD efficiently decarboxylated protocatechuate to catechol. We then integrated the Hpe pathway of Rhodococcus rhodochrous GD02 to enable acetovanillone conversion. Finally, deletion of catB enabled muconic acid accumulation. Our biocatalyst, strain RHAAL14, transformed the LDACs derived from the oxidation of softwood kraft lignin to muconic acid with a 97% molar yield and a titer of 1.4 g/L. The iterative, integrated metabolic engineering strategies described in this work advance the development of rhodococcal strains for microbial cell factories.
Anne T. Lalande, Logan D. Robeck, Dillon T. Hofsommer et al.· Metabolic Engineering· 1 citation
Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its toxicity limits growth and carbon-use efficiency at process-relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome-reduced strain of Pseudomonas putida and combined whole-genome sequencing, reverse engineering, transcriptomics, proteomics, and 13C-acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recovered most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi-omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism, and oxidative stress-associated proteins, indicating resource reallocation away from costly stress and non-essential programs. Fluxomics further revealed reduced EDEMP cycling and increased glyoxylate shunt flux, consistent with improved acetate-carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource-efficiency phenotype and identify gacA and fabB as actionable targets for acetate-based bioproduction.
N. Gurdo, A. Srinivasan, Tommaso Tagliani et al.· bioRxiv· 0 citations
4-hydroxybenzoate (HBA) is an aromatic component that forms the basis of great number of specialty plastics and fine chemicals. In pursuit of a greener and sustainable production, this article described Pseudomonas sp. NGC7, which has been metabolically engineered for the production of HBA from a mixture of aromatic compounds from sugarcane bagasse fractionated by alkaline treatment. While biomass-derived saccharides will continue to be in use, alternative carbon feedstocks for bioproduction are also desirable. For this reason, our work focused on producing HBA from a complex mixture of aromatics derived from lignocellulosic biomass. Following a mild alkaline treatment of sugarcane bagasse, the extracts principally comprised an aromatic mixture of p-coumarate, ferulate, 4-hydroxybenzaldehyde, HBA, syringaldehyde, syringate, vanillin, and vanillate. An HBA hydroxylase gene (pobA) knockout of NGC7 could produce HBA while utilizing the alkaline extract as a carbon source. The regulator gene (ferR) associated with the degradation pathway of hydroxycinnamate derivatives in NGC7 was identified, and its disruption enhanced the production rate of HBA from the extract. The engineered ⊿pobA⊿ferR strain proliferated and produced >6 g/L HBA with a yield of exceeding 90 mol% efficiently from the alkaline extract in the absence of saccharides. This study proved that "glucose-free HBA production" is attainable through metabolic flux manipulation in NGC7.
Yue-Ling Wang, Zen Ookawa, Yudai Higuchi et al.· Bioresource Technology· 0 citations
ABSTRACT Lactic acid production by Lachancea thermotolerans offers a promising biological strategy to restore wine acidity often compromised by climate change, yet this metabolic trait remains highly variable and poorly controlled. This study systematically evaluated the impact of controlled oxygenation regimes on fermentation performance and primary metabolism in three genetically distinct L. thermotolerans strains. Oxygen availability drastically altered metabolic fluxes. Under continuous aerobic conditions, carbon was redirected toward biomass formation (9- to 13-fold increase), with reduced ethanol and lactic acid yields, as well as increased succinic acid, acetoin, and 2,3-butanediol, whereas fully anaerobic conditions lowered fermentation performance but substantially increased lactic acid. These results confirmed that L. thermotolerans is a weakly Crabtree-positive yeast with mixed respiratory-fermentative metabolism. Carbon redistribution under aerobic conditions was not fully compensated by measured metabolites, resulting in substantially decreased carbon recovery (~70%) and suggesting undetermined respiratory CO2 production alongside diversion of carbon toward undetermined intracellular components. This imbalance was supported by a decline in cell density following peak growth, consistent with potential redox imbalance or oxidative stress. Lactic acid production occurred primarily under oxygen limitation, independent of ethanol formation, indicating a regulated, facultative adaptive response rather than a constitutive fermentation product. Strain-dependent differences further revealed distinct carbon allocation strategies. While oxygen strongly influenced metabolic behavior, pulse strategies showed limited, condition-dependent effects, highlighting the importance of timing and intensity for modulating lactic acid production. Overall, oxygen is a powerful lever to modulate metabolic fluxes in L. thermotolerans, but its application for predictable bio-acidification requires further optimization. IMPORTANCE Wine acidity is increasingly difficult to maintain under climate change, creating a need for reliable biological solutions. Some strains of Lachancea thermotolerans can produce lactic acid during fermentation, but this metabolic trait is inconsistent and difficult to control. This study shows that oxygen availability is a key factor shaping how this yeast uses carbon, shifting it between growth and lactic acid production. Under the continuous aerobic condition, carbon flux was redirected toward biomass, whereas the fully anaerobic condition favored lactic acid production but reduced fermentation performance. Importantly, pulsed oxygen additions did not provide consistent control of lactic acid production, but they revealed trends that highlight the importance of timing and intensity. These findings show that lactic acid production is a flexible response to environmental conditions rather than a constitutive trait. This work provides a foundation for developing strategies to better control lactic acid production through timed oxygen addition during fermentation. Wine acidity is increasingly difficult to maintain under climate change, creating a need for reliable biological solutions. Some strains of Lachancea thermotolerans can produce lactic acid during fermentation, but this metabolic trait is inconsistent and difficult to control. This study shows that oxygen availability is a key factor shaping how this yeast uses carbon, shifting it between growth and lactic acid production. Under the continuous aerobic condition, carbon flux was redirected toward biomass, whereas the fully anaerobic condition favored lactic acid production but reduced fermentation performance. Importantly, pulsed oxygen additions did not provide consistent control of lactic acid production, but they revealed trends that highlight the importance of timing and intensity. These findings show that lactic acid production is a flexible response to environmental conditions rather than a constitutive trait. This work provides a foundation for developing strategies to better control lactic acid production through timed oxygen addition during fermentation.
T. Jacobs, M. Setati, C. Camarasa et al.· Applied and Environmental Mi...· 0 citations
Industrial wastewaters-particularly those from the printing and dyeing sector-contain complex mixtures of recalcitrant nitrogen-containing organic compounds and microplastics that resist conventional treatment. Here we report a previously undescribed oxidase-protease fusion enzyme (A20674), discovered through transcriptomic analysis of wastewater-acclimated Chlorella vulgaris. Domain dissection reveals that the oxidase-like region drives broad-spectrum removal of N-heterocyclic and aromatic compounds, while the protease-like domain removes organic nitrogen. Engineered overexpression of this enzyme boosted organic nitrogen removal up to fifty-five-fold (final concentration 13-100 mg/L) across different industrial wastewaters. Capitalizing on this metabolic specialization, we constructed a synthetic microalgal-bacterial consortium in which Pseudomonas putida uses microalgal extracellular polysaccharides (EPS) as a carbon source to sustain growth, while supplying indole-3-acetic acid that stimulates microalgal biomass and EPS production. Reciprocal engineering of EPS overproduction in C. vulgaris and enhanced polysaccharide-catabolism in P. putida amplified this mutualistic loop. An evolved, PETase/MHETase-expressing P. putida strain simultaneously acquired elevated IAA output, further strengthening the symbiosis. The optimized consortium reduced organic nitrogen concentrations by sixteen-fold to discharge-compliant levels (≤5 mg/L), enhanced removal of recalcitrant organic nitrogen compounds by five-fold (final concentration 34 mg/L), and improved PET microplastic removal by nine-fold (initial concentration 1 g/L and final concentration 684 mg/L) in printing and dyeing wastewater. These findings uncover a bifunctional enzyme architecture for degrading structurally diverse industrial pollutants, and establish a synthetic-ecology framework for integrated removal of dissolved nitrogen-containing organic compounds and particulate microplastics-a combination unattainable by any single organism or conventional treatment process.
Rui Xiao, Yu-Shu Li, Peng Liu et al.· Bioresource Technology· 0 citations
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