-
In Vivo (Athens, Greece) Jun 2020The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other... (Review)
Review
The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.
Topics: Administration, Oral; Antiviral Agents; Bacterial Proteins; Betacoronavirus; COVID-19; Carbon-Sulfur Lyases; Clinical Trials as Topic; Coronavirus Infections; Cytokine Release Syndrome; Humans; Lymphocyte Activation; Macrophage Activation; Meta-Analysis as Topic; Methionine; Methylation; Pandemics; Pneumonia, Viral; Pseudomonas putida; RNA Caps; RNA Processing, Post-Transcriptional; RNA, Viral; Recombinant Proteins; S-Adenosylmethionine; SARS-CoV-2; T-Lymphocyte Subsets; Virus Replication
PubMed: 32503816
DOI: 10.21873/invivo.11948 -
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 -
Biotechnology For Biofuels and... Dec 2022With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the...
With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C) and epi-isozizaene (C) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing L-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass.
PubMed: 36510293
DOI: 10.1186/s13068-022-02235-6 -
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 -
Current Research in Food Science 2021is widely recognized as a spoiler of fresh foods under cold storage, and recently associated also with infections in clinical settings. The presence of antibiotic...
is widely recognized as a spoiler of fresh foods under cold storage, and recently associated also with infections in clinical settings. The presence of antibiotic resistance genes (ARGs) could be acquired and transmitted by horizontal genetic transfer and further increase the risk associated with its persistence in food and the need to be deeper investigated. Thus, in this work we presented a genomic and phenotypic analysis of the psychrotrophic ITEM 17297 to provide new insight into AR mechanisms by this species until now widely studied only for its spoilage traits. ITEM 17297 displayed resistance to several classes of antibiotics and it also formed huge amounts of biofilm; this latter registered increases at 15 °C in comparison to the optimum growth condition (30 °C). After ITEM 17297 biofilms exposure to antibiotic concentrations higher than 10-fold their MIC values no eradication occurred; interestingly, biomasses of biofilm cultivated at 15 °C increased their amount in a dose-dependent manner. Genomic analyses revealed determinants (RND-systems, ABC-transporters, and MFS-efflux pumps) for multi-drugs resistance (β-lactams, macrolides, nalidixic acid, tetracycline, fusidic acid and bacitracin) and a novel C allele. Biofilm and motility related pathways were depicted underlying their contribution to AR. Based on these results, underestimated psychrotrophic pseudomonas, such as the herein studied ITEM 17297 strain, might assume relevance in relation to the risk associated with the transfer of antimicrobial resistance genes to humans through cold stored contaminated foods. biofilm and AR related molecular targets herein identified will provide a basis to clarify the interaction between AR and biofilm formation and to develop novel strategies to counteract the persistence of multidrug resistant in the food chain.
PubMed: 33718885
DOI: 10.1016/j.crfs.2021.02.001 -
Microbial Biotechnology Sep 2019Implementation of single-stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically...
Implementation of single-stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking conspicuous phenotypes. Fortunately, the use of CRISPR/Cas9 as a device for counterselection of wild-type sequences helps to overcome this limitation. Merging ssDNA recombineering with CRISPR/Cas9 thus enables a suite of genomic edits with a straightforward approach: a CRISPR plasmid provides the spacer DNA sequence that directs the Cas9 nuclease ribonucleoprotein complex to cleave the genome at the wild-type sequences that have not undergone the change entered by the mutagenic ssDNA oligonucleotide(s). This protocol describes a complete workflow of the method optimized for P. putida, although it could in principle be applicable to many other pseudomonads. As an example, we show the deletion of the edd gene that encodes one key enzyme that operates the EDEMP cycle for glucose metabolism in P. putida EM42. By combining two incompatible CRISPR plasmids with different antibiotic selection markers, we show that the procedure can be cycled to implement consecutive deletions in the same strain, e.g. deletion of the pyrF gene following that of the edd mutant. This approach adds to the wealth of genetic technologies available for P. putida and strengthens its status as a chassis of choice for a suite of biotechnological applications.
Topics: CRISPR-Associated Protein 9; Clustered Regularly Interspaced Short Palindromic Repeats; DNA, Bacterial; DNA, Single-Stranded; Gene Editing; Genetics, Microbial; Pseudomonas putida; Recombination, Genetic
PubMed: 31237429
DOI: 10.1111/1751-7915.13453 -
Medicine Nov 2019The aim of this study was to analyze the risk factors, clinical features, and antimicrobial resistance of Pseudomonas putida (P putida) isolated from Tongji Hospital in... (Observational Study)
Observational Study
The aim of this study was to analyze the risk factors, clinical features, and antimicrobial resistance of Pseudomonas putida (P putida) isolated from Tongji Hospital in Wuhan, China.The data of 44 patients with P putida infections were retrospectively reviewed in this study. All cases of P putida strains were detected by the clinical laboratory of Tongji Hospital in the period of January 2010 to December 2017. Antimicrobial susceptibility testing was conducted using Kirby-Bauer method.Forty-four effective strains of P putida were isolated, including 32 inpatients and 12 outpatients. The 32 inpatients cases were obtained from various departments, which were urosurgery wards (n = 5, 15.6%), pediatrics wards (n = 4, 12.5%), hepatic surgery wards (n = 4, 12.5%), among others. The isolates had been discovered from urine specimens (28.2%), blood specimens (21.9%), sputum specimens (12.5%), and so on. Twenty-five patients had histories of catheterization before the isolation of P putida. Twenty-four patients were in immunocompromised states, 5 patients had undergone surgery, catheterization and were taking immunosuppressive therapy simultaneously. Polymicrobial infections were found in some P putida cases, especially Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Escherichia coli. All the patients had treated by antimicrobial before culture. Multi-drug-resistant strains were detected in 75% of P putida isolates. The P putida strains were resistant to trimethoprim/sulfamethoxazole (97.7%), aztreonam (88.6%), minocyline (74.3%), ticarcillin/clavulanic acid (72.7%), and sensitive to amikacin (86.4%), imipenem (62.8%), gentamicin (56.8%).Catheterization or other invasive procedures, immunocompromised states, and underlying diseases increased the risks of P putida infections. Moreover, the P putida strains were highly resistant to trimethoprim/sulfamethoxazole, aztreonam, minocyline, ticarcillin/clavulanic acid.
Topics: Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Child; Child, Preschool; China; Drug Resistance, Bacterial; Female; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Pseudomonas Infections; Pseudomonas putida; Retrospective Studies; Risk Factors; Young Adult
PubMed: 31689866
DOI: 10.1097/MD.0000000000017812 -
Heliyon Oct 2022Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by and and prevalent in food and feed. Microbial degradation is a promising strategy which can...
Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by and and prevalent in food and feed. Microbial degradation is a promising strategy which can be performed in mild and environmental friendly condition. This work is a step towards identifying the enzyme responsible for biodegradation of AFB1 by . Experiments were performed with lysate and compared with commercial lipase to see the degradation efficiency and the temperature stability. The cell free lysate of efficiently degraded AFB1 in a range of temperature from 20 to 90 °C. The lysate is thermostable and could retain its activity on pre-incubation up to 90 °C. Highest rate of degradation was observed at 70 °C. These observations show that the lysate is not only stable at higher temperatures but its enzymatic activity increases after incubation. Similarly, the commercial lipase degraded AFB1 efficiently. However, both, the lysate and lipase ceased degradation in presence of a lipase inhibitor, HgCl. The Hill function accurately predicted enzyme activity at various times and temperatures. Like lipase, the lysate also hydrolyses the p-nitrophenyl palmitate to p-nitrophenol. Kinetic parameters such as V, K and n values are good measures to characterize the lysate response with respect to changing paranitro phenyl palmitate levels. The substrate specificity test of lipase showed linear correlation between the absorbance at 410 nm vs amount of product paranitro phenol. The value of Km, Vmax and n are 0.62 mM, 355.7 μmol min and 1.29, respectively. The lipase gene presence in was confirmed using PCR technique. These observations indicate that the main enzyme responsible for AFB1 degradation by is lipase. Thus, lipase as a multifunctional biocatalyst provides a promising future for a variety of industries and may also help to ensure the food safety by degrading the mycotoxins.
PubMed: 36217476
DOI: 10.1016/j.heliyon.2022.e10809 -
ELife Nov 2019Microscopic water films allow bacteria to survive the seemingly dry surface of plant leaves.
Microscopic water films allow bacteria to survive the seemingly dry surface of plant leaves.
Topics: Plant Leaves; Pseudomonas fluorescens
PubMed: 31674912
DOI: 10.7554/eLife.52123 -
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