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Journal of Applied Microbiology Jul 2021To isolate, identify and characterize phenolic acid-degrading bacteria and reduce plant growth inhibition caused by phenolic acids.
AIMS
To isolate, identify and characterize phenolic acid-degrading bacteria and reduce plant growth inhibition caused by phenolic acids.
METHODS AND RESULTS
A total of 11 bacterial isolates with high phthalic acid (PA)-degrading ability were obtained using mineral salt medium (MSM) medium containing PA as sole carbon source. These isolates were identified as Arthrobacter globiformis, Pseudomonas putida and Pseudomonas hunanensis by sequence analyses of the 16S rRNA gene. Among them, five Pseudomonas strains could also effectively degrade ferulic acid (FA), p-hydroxybenzoic acid (PHBA) and syringic acid (SA) in MSM solution. P. putida strain 7 and P. hunanensis strain 10 showed highly efficient degradation of PA, SA, FA and PHBA, and could reduce their inhibition of lily, watermelon, poplar and strawberry seedling growth in soils respectively. These two strains could promote plant growth in soil with phenolic acids.
CONCLUSIONS
In this study, bacterial strains with highly efficient phenolic acid-degrading abilities could not only effectively reduce the autotoxicity of phenolic acids on plants but also were able to promote plant growth in soil with phenolic acids.
SIGNIFICANCE AND IMPACT OF THE STUDY
In this study, Pseudomonas can promote plant growth while degrading phenolic acids. Our results provide new choices for the biological removal of autotoxins.
Topics: Arthrobacter; Biodegradation, Environmental; Coumaric Acids; Gallic Acid; Hydroxybenzoates; Parabens; Phylogeny; Plant Development; Pseudomonas; Pseudomonas putida; RNA, Ribosomal, 16S; Rhizosphere; Seedlings; Soil Microbiology
PubMed: 33270328
DOI: 10.1111/jam.14956 -
Journal of Microbiology and... Apr 2023Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production...
Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an strain and was found to be non-functional. When the host of the LA biosensor was switched from to KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.
Topics: Bacterial Proteins; Escherichia coli; Pseudomonas putida; Biosensing Techniques
PubMed: 36775859
DOI: 10.4014/jmb.2301.01021 -
Environmental Pollution (Barking, Essex... Feb 2021Antibiotics are frequently used for clinical treatment and by the farming industry, and most of these are eventually released into the surrounding environment. The...
Antibiotics are frequently used for clinical treatment and by the farming industry, and most of these are eventually released into the surrounding environment. The impact of these antibiotic pollutants on environmental microorganisms is a concern. The present study showed that after Pseudomonas putida entered the logarithmic growth phase, tetracycline strongly stimulated its biofilm formation in a dose-dependent manner. This was supported by the increased expression of the key adhesin gene lapA in response to tetracycline treatment. Tetracycline treatment also changed the expression levels of the exopolysaccharide gene clusters alg, bcs and pea and the adhesin gene lapF. However, these genes did not participate in the tetracycline-induced biofilm formation. When a biofilm had been established, the P. putida population became more tolerant to tetracycline. Confocal laser scanning microscopic images showed that the interior of the biofilm provided favorable conditions that protected bacterial cells from tetracycline. Besides, biofilm formation of P. putida was also promoted by several other antibiotics, including oxytetracycline, fluoroquinolones, rifampicin, and imipenem, but not aminoglycosides. Susceptibility tests suggested that biofilm conferred a higher tolerance on P. putida cells to specific antibiotics (e.g., tetracyclines and fluoroquinolones). These antibiotics exerted a stronger inducing effect on biofilm formation. Together, our results indicate that P. putida actively forms robust biofilms in response to antibiotic stress, and the biofilms improve the survival of bacterial population under such stress.
Topics: Adhesins, Bacterial; Anti-Bacterial Agents; Biofilms; Pseudomonas putida
PubMed: 33359874
DOI: 10.1016/j.envpol.2020.116261 -
Microbial Biotechnology Nov 2021Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient...
Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.
Topics: Benzylamines; Lignin; Metabolic Engineering; Pseudomonas putida
PubMed: 33533574
DOI: 10.1111/1751-7915.13764 -
ACS Synthetic Biology May 2020FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). S12 is promising for FDCA production, but generating stable S12 is...
FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). S12 is promising for FDCA production, but generating stable S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO and CaCO to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating and (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable S12 strains for FDCA production.
Topics: Aldehyde Reductase; Calcium Carbonate; Chromatography, High Pressure Liquid; Clustered Regularly Interspaced Short Palindromic Repeats; Dicarboxylic Acids; Furans; Gene Dosage; Gene Editing; Manganese Compounds; Metabolic Engineering; Oxides; Oxidoreductases; Plasmids; Pseudomonas putida
PubMed: 32298581
DOI: 10.1021/acssynbio.0c00006 -
Current Opinion in Biotechnology Oct 2020
Review
Topics: Biological Products; Metabolic Engineering; Pseudomonas putida
PubMed: 32498036
DOI: 10.1016/j.copbio.2020.03.007 -
Microbial Biotechnology Jun 2024Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss...
Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.
Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.
Topics: Pseudomonas putida; Catabolite Repression; Pyruvate Dehydrogenase Complex; Hydrocarbons, Aromatic; Biodegradation, Environmental; Acetyl Coenzyme A; Pyruvic Acid; Gene Deletion; Metabolic Networks and Pathways
PubMed: 38923400
DOI: 10.1111/1751-7915.14514 -
Environmental Microbiology Aug 2022Indole-3-acetic acid (IAA) is the main naturally occurring auxin and is produced by organisms of all kingdoms of life. In addition to the regulation of plant growth and...
Indole-3-acetic acid (IAA) is the main naturally occurring auxin and is produced by organisms of all kingdoms of life. In addition to the regulation of plant growth and development, IAA plays an important role in the interaction between plants and growth-promoting and phytopathogenic bacteria by regulating bacterial gene expression and physiology. We show here that an IAA metabolizing plant-associated Pseudomonas putida isolate exhibits chemotaxis to IAA that is independent of auxin metabolism. We found that IAA chemotaxis is based on the activity of the PcpI chemoreceptor and heterologous expression of pcpI conferred IAA taxis to different environmental and human pathogenic isolates of the Pseudomonas genus. Using ligand screening, microcalorimetry and quantitative chemotaxis assays, we found that PcpI failed to bind IAA directly, but recognized and mediated chemoattractions to various aromatic compounds, including the phytohormone salicylic acid. The expression of pcpI and its role in the interactions with plants was also investigated. PcpI extends the range of central signal molecules recognized by chemoreceptors. To our knowledge, this is the first report on a bacterial receptor that responds to two different phytohormones. Our study reinforces the multifunctional role of IAA and salicylic acid as intra- and inter-kingdom signal molecules.
Topics: Chemotaxis; Humans; Indoleacetic Acids; Plant Growth Regulators; Plants; Pseudomonas putida; Salicylic Acid
PubMed: 35088505
DOI: 10.1111/1462-2920.15920 -
Metabolic Engineering Mar 2023Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the...
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
Topics: Pseudomonas putida; 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Mutagenesis; Fatty Acids
PubMed: 36796578
DOI: 10.1016/j.ymben.2023.02.006 -
Biotechnology Progress May 2022The red pigment prodigiosin is of high pharmaceutical interest, due to its potential applications as an antitumor drug and antibiotic agent. As previously demonstrated,...
The red pigment prodigiosin is of high pharmaceutical interest, due to its potential applications as an antitumor drug and antibiotic agent. As previously demonstrated, Pseudomonas putida KT2440 is a suitable host for prodigiosin production, as it exhibits high tolerance toward the antimicrobial properties of prodigiosin. So far, prodigiosin concentrations of up to 94 mg/L have been achieved in shake flask cultivations. For the characterization and optimization of the prodigiosin production process, the scattered light of P. putida and fluorescence of prodigiosin was measured. The excitation and emission wavelengths for prodigiosin measurement were analyzed by recording 2D fluorescence spectra. The strongest prodigiosin fluorescence was obtained at a wavelength combination of 535/560 nm. By reducing the temperature to 18 °C and using 16 g/L glucose, the prodigiosin concentration was more than doubled compared with the initial cultivation conditions. The obtained results demonstrate the capabilities of parallelized microscale cultivations combined with noninvasive online monitoring of fluorescence for rapid bioprocess development, using prodigiosin as a molecule of current biotechnological interest.
Topics: Anti-Bacterial Agents; Fluorescence; Glucose; Prodigiosin; Pseudomonas putida
PubMed: 35170260
DOI: 10.1002/btpr.3245