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Frontiers in Bioengineering and... 2023Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored...
Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from and demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaC synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in KT2440.
PubMed: 38026847
DOI: 10.3389/fbioe.2023.1275036 -
Bioelectrochemistry (Amsterdam,... Jun 2024Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox...
Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox balances. This electron discharge is also attractive to be used for bioelectrochemical applications. However, elements of the respiratory pathways that interact with phenazines are not well understood. Five terminal oxidases are involved in the aerobic electron transport chain (ETC) of Pseudomonas putida and P. aeruginosa. The latter bacterium also includes four reductases that allow for denitrification. Here, we explored if phenazine-1-carboxylic acid interacts with those elements to enhance anodic electron discharge and drive bacterial growth in oxygen-limited conditions. Bioelectrochemical evaluations of terminal oxidase-deficient mutants of both Pseudomonas strains and P. aeruginosa with stimulated denitrification pathways indicated no direct beneficial interaction of phenazines with ETC elements for extracellular electron discharge. However, the single usage of the Cbb3-2 oxidase increased phenazine production, electron discharge, and cell growth. Assays with purified periplasmic cytochromes NirM and NirS indicated that pyocyanin acts as their electron donor. We conclude that phenazines play an important role in electron transfer to, between, and from terminal oxidases under oxygen-limiting conditions and their modulation might enhance EET. However, the phenazine-anode interaction cannot replace oxygen respiration to deliver energy for biomass formation.
Topics: Electron Transport; Pseudomonas aeruginosa; Pseudomonas putida; Electrons; Phenazines; Oxygen
PubMed: 38181591
DOI: 10.1016/j.bioelechem.2023.108636 -
Journal of Agricultural and Food... Jul 2023Owing to their physiological activities, plant-derived phenolic acids, such as protocatechuic acid (PCA), have extensive applications and market prospects. However,...
Owing to their physiological activities, plant-derived phenolic acids, such as protocatechuic acid (PCA), have extensive applications and market prospects. However, traditional production processes present numerous challenges and cannot meet increasing market demands. Hence, we aimed to biosynthesize PCA by constructing an efficient microbial factory via metabolic engineering of KT2440. Glucose metabolism was engineered by deleting the genes for gluconate 2-dehydrogenase to enhance PCA biosynthesis. To increase the biosynthetic metabolic flux, one extra copy of the genes , , and was inserted into the genome. The resultant strain, KGVA04, produced 7.2 g/L PCA. By inserting the degradation tags GSD and DAS to decrease the amount of shikimate dehydrogenase, PCA biosynthesis was increased to 13.2 g/L in shake-flask fermentation and 38.8 g/L in fed-batch fermentation. To the best of our knowledge, this was the first use of degradation tags to adjust the amount of a key enzyme at the protein level in KT2440, evidencing the remarkable potential of this method for naturally producing phenolic acids.
Topics: Pseudomonas putida; Metabolic Engineering; Hydroxybenzoates
PubMed: 37365996
DOI: 10.1021/acs.jafc.3c01511 -
Biotechnology For Biofuels and... Apr 2024Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries...
BACKGROUND
Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries offer eco-friendly alternatives. The solvent-tolerant Pseudomonas putida DOT-T1E serves as a suitable host for producing aromatic compounds, specifically L-phenylalanine and its derivative, 2-phenylethanol (2-PE), which find widespread applications in various industries.
RESULTS
This study focuses on enhancing 2-PE production in two L-phenylalanine overproducing strains of DOT-T1E, namely CM12-5 and CM12-5Δgcd (xylABE), which grow with glucose and glucose-xylose, respectively. To synthesize 2-PE from L-phenylalanine, these strains were transformed with plasmid pPE-1, bearing the Ehrlich pathway genes, and it was found higher 2-PE production with glucose (about 50-60 ppm) than with xylose (< 3 ppm). To understand the limiting factors, we tested the addition of phenylalanine and intermediates from the Ehrlich and shikimate pathways. The results identified intracellular L-phenylalanine as a key limiting factor for 2-PE production. To overcame this limitation, a chorismate mutase/prephenate dehydratase variant-insentive to feedback inhibition by aromatic amino acids-was introduced in the producing strains. This led to increased L-phenylalanine production and subsequently produced more 2-PE (100 ppm). Random mutagenesis of the strains also produced strains with higher L-phenylalanine titers and increased 2-PE production (up to 120 ppm). The improvements resulted from preventing dead-end product accumulation from shikimate and limiting the catabolism of potential pathway intermediates in the Ehrlich pathway. The study explored agricultural waste substrates, such as corn stover, sugarcane straw and corn-syrup as potential C sources. The best results were obtained using 2G substrates at 3% (between 82 and 100 ppm 2-PE), with glucose being the preferred sugar for 2-PE production among the monomeric sugars in these substrates.
CONCLUSIONS
The findings of this study offer strategies to enhance phenylalanine production, a key substrate for the synthesis of aromatic compounds. The ability of P. putida DOT-T1E to thrive with various C-sources and its tolerance to substrates, products, and potential toxicants in industrial wastes, are highlighted. The study identified and overcome possible bottlenecks for 2-PE production. Ultimately, the strains have potential to become efficient microbial platforms for synthesizing 2-PE from agro-industrial waste materials.
PubMed: 38566218
DOI: 10.1186/s13068-024-02498-1 -
Advances in Experimental Medicine and... Mar 2024Beyond their role as protein-building units, amino acids are modulators of multiple behaviours in different microorganisms. In the root-colonizing beneficial bacterium...
Beyond their role as protein-building units, amino acids are modulators of multiple behaviours in different microorganisms. In the root-colonizing beneficial bacterium Pseudomonas putida (recently proposed to be reclassified as alloputida) KT2440, current evidence suggests that arginine functions both as a metabolic indicator and as an environmental signal molecule, modulating processes such as chemotactic responses, siderophore-mediated iron uptake or the levels of the intracellular second messenger cyclic diguanylate (c-di-GMP). Using microcalorimetry and extracellular flux analysis, in this work we have studied the metabolic adaptation of P. putida KT2440 to the presence of L-arginine in the growth medium, and the influence of mutations related to arginine metabolism. Arginine causes rapid changes in the respiratory activity of P. putida, particularly magnified in a mutant lacking the transcriptional regulator ArgR. The metabolic activity of mutants affected in arginine transport and metabolism is also altered during biofilm formation in the presence of the amino acid. The results obtained here further support the role of arginine as a metabolic signal in P. putida and the relevance of ArgR in the adaptation to the amino acid. They also serve as proof of concept on the use of calorimetric and extracellular flux techniques to analyse metabolic responses in bacteria and the impact of different mutant backgrounds on such responses.
PubMed: 38429473
DOI: 10.1007/5584_2024_797 -
Environmental Research Nov 2023Effluent from biogas plants can contribute to serious environmental issues due to its high organic compound content and its dark color. This study utilizes Pseudomonas...
Effluent from biogas plants can contribute to serious environmental issues due to its high organic compound content and its dark color. This study utilizes Pseudomonas putida strains isolated from activated sludge in a wastewater treatment plant to reduce the organic compound content and dark color of biogas plant effluent. The treatment of effluent from the anaerobic digestion of distillery wastewater with P. putida in the best evaluated conditions (sample concentration of 50% v/v, agitation and aeration rates of 250 rpm and 1.0 vvm for 6 days) produced significant decolorization and organic removal efficacies of 34.29 ± 0.87% and 38.94 ± 0.63%, respectively. Bioremediation efficiency was dependent on bacterial growth, and the organic content and dissolved oxygen in the sample during cultivation were key factors in bacterial growth. The naturally selected bacterium could tolerate relatively high levels of organic content and work synergistically with other indigenous microorganisms found in the biogas plant effluent. Therefore, the bioremediation of biogas plant effluent using naturally selected P. putida can feasibly be applied in biogas power plants where sterilization is not necessary.
Topics: Wastewater; Waste Disposal, Fluid; Pseudomonas putida; Biofuels; Sewage; Bioreactors; Anaerobiosis
PubMed: 37532210
DOI: 10.1016/j.envres.2023.116807 -
Science Advances Nov 2023Cross-linked elastomers are stretchable materials that typically are not recyclable or biodegradable. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are soft and...
Cross-linked elastomers are stretchable materials that typically are not recyclable or biodegradable. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are soft and ductile, making these bio-based polymers good candidates for biodegradable elastomers. Elasticity is commonly imparted by a cross-linked network structure, and covalent adaptable networks have emerged as a solution to prepare recyclable thermosets via triggered rearrangement of dynamic covalent bonds. Here, we develop biodegradable and recyclable elastomers by chemically installing the covalent adaptable network within biologically produced mcl-PHAs. Specifically, an engineered strain of was used to produce mcl-PHAs containing pendent terminal alkenes as chemical handles for postfunctionalization. Thiol-ene chemistry was used to incorporate boronic ester (BE) cross-links, resulting in PHA-based vitrimers. mcl-PHAs cross-linked with BE at low density (<6 mole %) affords a soft, elastomeric material that demonstrates thermal reprocessability, biodegradability, and denetworking at end of life. The mechanical properties show potential for applications including adhesives and soft, biodegradable robotics and electronics.
Topics: Polyhydroxyalkanoates; Pseudomonas putida; Elasticity; Elastomers
PubMed: 37992173
DOI: 10.1126/sciadv.adi1735 -
Journal of Environmental Management Oct 2023Biosynthesis of silver nanoparticles (AgNPs) by plant extracts and its antibacterial utilization has attracted great attention due to the spontaneous reducing and...
Biosynthesis of silver nanoparticles (AgNPs) by plant extracts and its antibacterial utilization has attracted great attention due to the spontaneous reducing and capping capacities of phytochemicals. However, the preferential role and mechanisms of the functional phytochemicals from different plants on AgNPs synthesis, and its catalytic and antibacterial performance remain largely unknown. This study used three widespread arbor species, including Eriobotrya japonica (EJ), Cupressus funebris (CF) and Populus (PL), as the precursors and their leaf extracts as reducing and stabilizing agents for the biosynthesis of AgNPs. A total of 18 phytochemicals in leaf extracts were identified by ultra-high liquid-phase mass spectrometer. For EJ extracts, most kinds of flavonoids participated in the generation of AgNPs by a reduced content of 5∼10%, while for CF extracts, about 15∼40% of the polyphenols were consumed to reduce Ag to Ag. Notably, the more stable and homogeneous spherical AgNPs with smaller size (≈38 nm) and high catalytic capacity on Methylene blue were obtained from EJ extracts rather than CF extracts, and no AgNPs were synthesized from PL extracts, indicating that flavonoids are superior than polyphenols to act as reducer and stabilizer in AgNPs biosynthesis. The antibacterial activities against Gram-positive (Staphylococcus aureus and Bacillus mycoides) and Gram-negative bacteria (Pseudomonas putida and Escherichia coli) were higher in EJ-AgNPs than that in CF-AgNPs, which confirmed the synergistic antibacterial effects of flavonoids combined with AgNPs in EJ-AgNPs. This study provides a significant reference on the biosynthesis of AgNPs with efficient antibacterial utilization underlying effect of abundant flavonoids in plant extracts.
Topics: Metal Nanoparticles; Silver; Plant Extracts; Anti-Bacterial Agents; Polyphenols; Flavonoids; Spectroscopy, Fourier Transform Infrared; Microbial Sensitivity Tests
PubMed: 37418916
DOI: 10.1016/j.jenvman.2023.118546 -
International Journal of Antimicrobial... Oct 2023The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence...
OBJECTIVE
The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence activities such as biofilms, motility, energy-dependent efflux pumps, and β-lactamase activity, which are modulated primarily by quorum sensing (QS), defining their potential applications.
METHOD
The microdilution method was used to determine the minimum inhibitory concentration (MIC) and sub-inhibitory concentrations (SICs) of the extracts against reference pathogenic bacteria. The antibacterial mode of action was determined by labelling bacterial cells in in vivo cell-tracking experiments.
RESULTS
Antibiograms showed that PoPE inhibited bacteria at lower concentrations, and PePE had a stronger effect against Klebsiella pneumoniae. Both extracts caused significant cell membrane damage (CMD), whereas GrPE did not. At SICs, all extracts showed anti-QS activity, especially PePE, which inhibited violacein and pyocyanin production at 1/128 × MIC. Additionally, QS autoinducers found in Chromobacterium violaceum and Pseudomonas aeruginosa were modulated by the extracts; PePE showed the highest modulation. Antibiofilm assays revealed that GrPE, at MIC and 2 × MIC, acted as a potent antibiofilm agent against biofilms of Pseudomonas putida, Bacillus cereus, and Staphylococcus aureus, which was related to disruption of swarming motility by GrPE. All extracts, especially PoPE, exerted a potent effect against the activation of efflux pumps of P. aeruginosa as well as β-lactamase activity in K. pneumoniae.
CONCLUSION
Results suggest that the anti-virulence potential of the extracts may be related to their effect as extracellular autoinducer modulators. This study allowed to define potential applications of these extracts.
PubMed: 37517626
DOI: 10.1016/j.ijantimicag.2023.106937 -
Applied Microbiology and Biotechnology Aug 2023While the in situ return of corn straw can improve soil fertility and farmland ecology, additional bacterial agents are required in low-temperature areas of northern...
While the in situ return of corn straw can improve soil fertility and farmland ecology, additional bacterial agents are required in low-temperature areas of northern China to accelerate straw degradation. Moisture is an important factor affecting microbial activity; however, owing to a lack of bacterial agents adapted to low-temperature complex soil environments, the effects of soil moisture on the interaction between exogenous bacterial agents and indigenous soil microorganisms remain unclear. To this end, we explored the effect of the compound bacterial agent CFF constructed using Pseudomonas putida and Acinetobacter lwoffii, developed to degrade corn straw in low-temperature soils (15 °C), on indigenous bacterial and fungal communities under dry (10% moisture content), slightly wet (20%), and wet (30%) soil-moisture conditions. The results showed that CFF application significantly affected the α-diversity of bacterial communities and changed both bacterial and fungal community structures, enhancing the correlation between microbial communities and soil-moisture content. CFF application also changed the network structure and the species of key microbial taxa, promoting more linkages among microbial genera. Notably, with an increase in soil moisture, CFF enhanced the rate of corn straw degradation by inducing positive interactions between bacterial and fungal genera and enriching straw degradation-related microbial taxa. Overall, our study demonstrates the alteration of indigenous microbial communities using bacterial agents (CFF) to overcome the limitations of indigenous microorganisms for in situ straw-return agriculture in low-temperature areas. KEY POINTS: • Low-temperature and variable moisture conditions (10-30%) were compared • Soil microbial network structure and linkages between genera were altered • CFF improves straw degradation via positive interactions between soil microbes.
Topics: Zea mays; Temperature; Soil Microbiology; Agriculture; Soil; Bacteria
PubMed: 37392246
DOI: 10.1007/s00253-023-12644-8