Showing papers on "Pseudomonas putida published in 2020"
TL;DR: In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams.
Abstract: Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain - which contains all the genetic modifications detailed above - demonstrated a 53% and 200% increase in mcl-PHA titre (g l-1 ) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.
113 citations
TL;DR: This study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P. putida, which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas.
Abstract: Genome-scale reconstructions of metabolism are computational species-specific knowledge bases able to compute systemic metabolic properties. We present a comprehensive and validated reconstruction of the biotechnologically relevant bacterium Pseudomonas putida KT2440 that greatly expands computable predictions of its metabolic states. The reconstruction represents a significant reactome expansion over available reconstructed bacterial metabolic networks. Specifically, iJN1462 (i) incorporates several hundred additional genes and associated reactions resulting in new predictive capabilities, including new nutrients supporting growth; (ii) was validated by in vivo growth screens that included previously untested carbon (48) and nitrogen (41) sources; (iii) yielded gene essentiality predictions showing large accuracy when compared with a knock-out library and Bar-seq data; and (iv) allowed mapping of its network to 82 P. putida sequenced strains revealing functional core that reflect the large metabolic versatility of this species, including aromatic compounds derived from lignin. Thus, this study provides a thoroughly updated metabolic reconstruction and new computable phenotypes for P. putida, which can be leveraged as a first step toward understanding the pan metabolic capabilities of Pseudomonas.
101 citations
TL;DR: The recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory are summarized and Pseudomonas putida advances to a global industrial cell factory.
Abstract: Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. KEY POINTS: • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs.
95 citations
TL;DR: This work describes a versatile, robust and user‐friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3–5 days.
Abstract: Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large-scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user-friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3-5 days. The protocol presented here is based on DNA recombination forced by double-stranded DNA cuts (through the activity of the I-SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR-Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult-to-delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome.
88 citations
TL;DR: This work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.
68 citations
TL;DR: Three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways, were implemented to increase the substrate spectrum in Pseudomonas putida KT2440 to allow engineering of tailored chassis for valuable bioproduct synthesis.
Abstract: Pseudomonas putida KT2440 is a well-established chassis in industrial biotechnology. To increase the substrate spectrum, we implemented three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways. The synthetic operons contain genes from Escherichia coli and Pseudomonas taiwanensis. For isolating the Dahms pathway in P. putida KT2440 two genes (PP_2836 and PP_4283), encoding an endogenous enzyme of the Weimberg pathway and a regulator for glycolaldehyde degradation, were deleted. Before and after adaptive laboratory evolution, these strains were characterized in terms of growth and synthesis of mono-rhamnolipids and pyocyanin. The engineered strain using the Weimberg pathway reached the highest maximal growth rate of 0.30 h-1. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L-1 mono-rhamnolipids and 30 mg L-1 pyocyanin were reached by the evolved strain using the Weimberg or an engineered strain using the Isomerase pathway, respectively. The different stoichiometries of the three xylose utilization pathways may allow engineering of tailored chassis for valuable bioproduct synthesis.
68 citations
TL;DR: This work proposes a mechanism for extracellular nutrient acquisition from aromatic compounds by soil bacteria, which holds promise for improving the efficiency of microbial lignin conversion and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.
Abstract: Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.
68 citations
TL;DR: Adaptive evolution is employed to improve muconate production in strains incapable of producing 2-ketogluconate by P. putida in the context of gcd deletion and targeted engineering recapitulate improvements achieved by evolution.
65 citations
TL;DR: Pseudomonas putida YC-AE1 is reported as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration and exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions.
Abstract: Bisphenol A is an important organic chemical as an intermediate, final and inert ingredient in manufacturing of many important products like polycarbonate plastics, epoxy resins, flame retardants, food–drink packaging coating, and other. BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs. Bacterial degradation has been consider as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods. This study aimed to isolate and identify bacterial strain capable to degrade and tolerate high concentrations of this pollutant, studying the factors affecting the degradation process and study the degradation mechanism of this strain. YC-AE1 is a Gram negative bacterial strain isolated from soil and identified as Pseudomonas putida by 16S rRNA gene sequence and BIOLOG identification system. This strain found to have a high capacity to degrade the endocrine disruptor Bisphenol A (BPA). Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation and the results obtained by significant model were 7.2, 30 °C and 2.5% for optimum initial pH, temperature and inoculum size, respectively. Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA. Analysis of variance (ANOVA) showed high coefficient of determination, R2 and Adj-R2 which were 0.9979 and 0.9935, respectively. Substrate analysis found that, strain YC-AE1 could degrade a wide variety of bisphenol A-related pollutants such as bisphenol B, bisphenol F, bisphenol S, Dibutyl phthalate, Diethylhexyl phthalate and Diethyl phthalate in varying proportion. Pseudomonas putida YC-AE1 showed high ability to degrade a wide range of BPA concentrations (0.5–1000 mg l− 1) with completely degradation for 500 mg l− 1 within 72 h. Metabolic intermediates detected in this study by HPLC-MS were identified as 4,4-dihydroxy-alpha-methylstilbene, p-hydroxybenzaldeyde, p-hydroxyacetophenone, 4-hydroxyphenylacetate, 4-hydroxyphenacyl alcohol, 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl) propanoate. This study reports Pseudomonas putida YC-AE1 as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration. It exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions. Moreover, it degrades BPA in a short time via two different degradation pathways.
58 citations
TL;DR: This work has developed a toolbox for both target- and self-curing of plasmid DNA in Pseudomonas species, and quick genome engineering of P. putida using self- curing plasmids is demonstrated through genome reduction of the platform strain EM42 by eliminating all genes encoding β-lactamases, the catabolic ben gene cluster, and the pyoverdine synthesis machinery.
52 citations
TL;DR: A novel expression system based on the well‐known XylS/Pm transcriptional regulator/promoter pair from the soil bacterium Pseudomonas putida mt‐2, in which the key functional elements are physically decoupled, which constitutes an alternative tool to altogether suppress leaky gene expression.
Abstract: Most of the gene expression systems available for Gram-negative bacteria are afflicted by relatively high levels of basal (i.e. leaky) expression of the target gene(s). This occurrence affects the system dynamics, ultimately reducing the output and productivity of engineered pathways and synthetic circuits. In order to circumvent this problem, we have designed a novel expression system based on the well-known XylS/Pm transcriptional regulator/promoter pair from the soil bacterium Pseudomonas putida mt-2, in which the key functional elements are physically decoupled. By integrating the xylS gene into the chromosome of the platform strain KT2440, while placing the Pm promoter into a set of standard plasmid vectors, the inducibility of the system (i.e. the output difference between the induced and uninduced state) improved up to 170-fold. We further combined this modular system with an extra layer of post-translational control by means of conditional proteolysis. In this setup, the target gene is tagged with a synthetic motif dictating protein degradation. When the system features were characterized using the monomeric superfolder GFP as a model protein, the basal levels of fluorescence were brought down to zero (i.e. below the limit of detection). In all, these novel expression systems constitute an alternative tool to altogether suppress leaky gene expression, and they can be easily adapted to other vector formats and plugged-in into different Gram-negative bacterial species at the user's will.
TL;DR: A new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies is presented.
TL;DR: In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida by harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, and biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride.
Abstract: Fluorine is a key element in the synthesis of molecules broadly used in medicine, agriculture and materials. Addition of fluorine to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochemistry of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochemical reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chemical landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.
TL;DR: Functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the Pseudomonas putida bacterium is provided, providing a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.
Abstract: With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq) Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA) Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yieldsIMPORTANCE To engineer novel metabolic pathways into P putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired
TL;DR: A single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system is presented, which enables tunable, tightly controlled gene repression of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously.
Abstract: Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single-plasmid CRISPR-interference (CRISPRi) system expressing a nuclease-deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.
TL;DR: The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.
Abstract: Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation. In this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant. These desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.
TL;DR: A role of cyclodipeptides for bacterial phytostimulation is suggested in plant biomass production, lateral root formation, and activation of auxin signaling in Arabidopsis thaliana by Pseudomonas putida and P. fluorescence.
Abstract: Plant growth-promoting rhizobacteria modulate root development through different mechanisms. This work was conducted to evaluate the effects of root colonization by Pseudomonas putida and Pseudomonas fluorescens in biomass production, lateral root formation, and activation of auxin signaling in Arabidopsis thaliana. Selected strains of P. putida and P. fluorescens were tested for modification of DR5::uidA, BA3::uidA and HS::AXR3NT-GUS auxin-related gene expression, and to promote root hair and lateral root formation in WT and tir1-1, tir1-1afb2-1afb3-1, arf7-1, arf19-1, arf7-1arf19-1, and rhd6 mutants. Production of cyclodipeptides with possible roles in auxin signaling was also determined in P. putida and P. fluorescens culture supernatants by gas chromatography–mass spectrometry. P. putida and P. fluorescens stimulated lateral root and root hair formation and increased plant biomass, which correlated with an induction of the auxin response. Genetic analyses suggested that growth promotion involves auxin signaling as tir1-1, tir1-1afb2-1afb3-1, arf7-1, arf19-1, and arf7-1arf19-1 mutants showed decreased lateral root response to inoculation and because P. putida and P. fluorescens restored root hair development in the rhd6 mutant. It was also found that these bacteria produce the cyclodipeptides cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Phe), and cyclo(L-Pro-L-Tyr), which modulates auxin-responsive gene expression in roots. Our results suggest a role of cyclodipeptides for bacterial phytostimulation.
TL;DR: A deeper understanding of microbial 1,4-butanediol metabolism in P. putida enables the more efficient metabolism of these diols, thereby enabling biotechnological valorization of plastic monomers in a bio-upcycling approach.
Abstract: Plastics, in all forms, are a ubiquitous cornerstone of modern civilization. Although humanity undoubtedly benefits from the versatility and durability of plastics, they also cause a tremendous burden for the environment. Bio-upcycling is a promising approach to reduce this burden, especially for polymers that are currently not amenable to mechanical recycling. Wildtype P. putida KT2440 is able to grow on 1,4-butanediol as sole carbon source, but only very slowly. Adaptive laboratory evolution (ALE) led to the isolation of several strains with significantly enhanced growth rate and yield. Genome re-sequencing and proteomic analysis were applied to characterize the genomic and metabolic basis of efficient 1,4-butanediol metabolism. Initially, 1,4-butanediol is oxidized to 4-hydroxybutyrate, in which the highly expressed dehydrogenase enzymes encoded within the PP_2674-2680 ped gene cluster play an essential role. The resulting 4-hydroxybutyrate can be metabolized through three possible pathways: (i) oxidation to succinate, (ii) CoA activation and subsequent oxidation to succinyl-CoA, and (iii) beta oxidation to glycolyl-CoA and acetyl-CoA. The evolved strains were both mutated in a transcriptional regulator (PP_2046) of an operon encoding both beta-oxidation related genes and an alcohol dehydrogenase. When either the regulator or the alcohol dehydrogenase is deleted, no 1,4-butanediol uptake or growth could be detected. Using a reverse engineering approach, PP_2046 was replaced by a synthetic promotor (14g) to overexpress the downstream operon (PP_2047-2051), thereby enhancing growth on 1,4-butanediol. This work provides a deeper understanding of microbial 1,4-butanediol metabolism in P. putida, which is also expandable to other aliphatic alpha-omega diols. It enables the more efficient metabolism of these diols, thereby enabling biotechnological valorization of plastic monomers in a bio-upcycling approach.
TL;DR: It is deciphered that P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge.
Abstract: Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large-scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response-like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3-hydroxyalkanoates (3-HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4% of glucose uptake to build those 3-HA-based energy buffers. Concomitantly, genes that are related to amino acid catabolism and β-oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short- and long-term responses that increase the cellular maintenance by about 17% under the industrial-like conditions tested.
TL;DR: The successful engineering of Pseudomonas putida KT2440 strain for the direct biosynthesis of adipic acid from lignin-derived aromatics is reported, representing the first example of the direct adipic Acid production from model compounds of lignIn depolymerization.
05 Mar 2020
TL;DR: A genome editing strategy is developed for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-λ-Red system with pyrF as a selection marker that maintains cell viability and genetic stability, increases mutation efficiency, and simplifies genetic manipulation.
Abstract: Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. However, its recalcitrance and toxicity poses a challenge for ferulic acid-to-bioproducts bioconversion. Here, we develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-λ-Red system with pyrF as a selection marker, which maintains cell viability and genetic stability, increases mutation efficiency, and simplifies genetic manipulation. Via this method, four functional modules, comprised of nine genes involved in ferulic acid catabolism and polyhydroxyalkanoate biosynthesis, were integrated into the genome, generating the KTc9n20 strain. After metabolic engineering and optimization of C/N ratio, polyhydroxyalkanoate production was increased to ~270 mg/L, coupled with ~20 mM ferulic acid consumption. This study not only establishes a simple and efficient genome editing strategy, but also offers an encouraging example of how to apply this method to improve microbial aromatic compound bioconversion. Yueyue Zhou et al. develop a genetic engineering method that increases the production of polyhydroxyalkanoate from ferulic acid, which is toxic at high concentrations. This study provides insight into the bioconversion of the aromatic compound in Pseudomonas.
TL;DR: Endophytic colonization of rice triggered alteration in root morphology, possibly, due trade-off between growth and defense, and rice seedlings emerged from endophyte priming showed down-regulation of stress responsive OsACO4 and OsACS6 involved in inter-nodal elongation which plays an important role in growth and development.
TL;DR: It is shown that P. putida KT2440 can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization.
Abstract: New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria in agricultural practices. The non-pathogenic bacterium Pseudomonas putida KT2440 is an excellent root colonizer of crops of agronomical importance and has been shown to activate the induced systemic resistance of plants in response to certain foliar pathogens. In this work, we have analyzed additional plant growth promotion features of this strain. We show it can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization. Inoculation with P. putida KT2440 significantly improved seed germination and root and stem length of soybean and corn plants under saline conditions compared to uninoculated plants, whereas the effects were minor under non-saline conditions. Also, random transposon mutagenesis was used for preliminary identification of KT2440 genes involved in bacterial tolerance to saline stress. One of the obtained mutants was analyzed in detail. The disrupted gene encodes a predicted phosphoethanolamine-lipid A transferase (EptA), an enzyme described to be involved in the modification of lipid A during lipopolysaccharide (LPS) biosynthesis. This mutant showed changes in exopolysaccharide (EPS) production, low salinity tolerance, and reduced competitive fitness in the rhizosphere.
11 Mar 2020
TL;DR: The present study illustrated the decomposition mechanism of acephate under different conditions, and the same may be applied to the removal of other xenobiotic compounds.
Abstract: Many bacteria have the potential to use specific pesticides as a source of carbon, phosphorous, nitrogen and sulphur. Acephate degradation by microbes is considered to be a safe and effective method. The overall aim of the present study was to identify acephate biodegrading microorganisms and to investigate the degradation rates of acephate under the stress of humic acid and most common metal ions Fe(III) and copper Cu(II). Pseudomonas azotoformanss strain ACP1, Pseudomonas aeruginosa strain ACP2, and Pseudomonas putida ACP3 were isolated from acephate contaminated soils. Acephate of concentration 100 ppm was incubated with separate strain inoculums and periodic samples were drawn for UV—visible, FTIR (Fourier-transform infrared spectroscopy) and MS (Mass Spectrometry) analysis. Methamidophos, S-methyl O-hydrogen phosphorothioamidate, phosphenothioic S-acid, and phosphenamide were the major metabolites formed during the degradation of acephate. The rate of degradation was applied using pseudo-first-order kinetics to calculate the half-life (t1/2) values, which were 14.33–16.72 d−1 (strain(s) + acephate), 18.81–21.50 d−1 (strain(s) + acephate + Cu(II)), 20.06 –23.15 d−1 (strain(s) + acephate + Fe(II)), and 15.05–17.70 d−1 (strains + acephate + HA). The biodegradation efficiency of the three bacterial strains can be ordered as P. aeruginosa > P. putida > P. azotoformans. The present study illustrated the decomposition mechanism of acephate under different conditions, and the same may be applied to the removal of other xenobiotic compounds.
TL;DR: It is identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida, and deletion of the second step in coumarate catabolism resulted in increased production of the type III polyketide bisdemethoxycurcumin.
TL;DR: The synergistic physical-biological treatment presented herein is expected to have great potential in the field of wastewater treatment.
TL;DR: A holin family bacteriocin and a mitomycin-like biosynthetic protein were found to be core-specific for P. cholororaphis and it is hypothesized that these proteins may confer a competitive advantage against other root-colonizers.
Abstract: The Pseudomonas genus includes many species living in diverse environments and hosts. It is important to understand which are the major evolutionary groups and what are the genomic/proteomic components they have in common or are unique. Towards this goal, we analyzed 494 complete Pseudomonas proteomes and identified 297 core-orthologues. The subsequent phylogenomic analysis revealed two well-defined species (Pseudomonas aeruginosa and Pseudomonas chlororaphis) and four wider phylogenetic groups (Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas putida) with a sufficient number of proteomes. As expected, the genus-level core proteome was highly enriched for proteins involved in metabolism, translation, and transcription. In addition, between 39–70% of the core proteins in each group had a significant presence in each of all the other groups. Group-specific core proteins were also identified, with P. aeruginosa having the highest number of these and P. fluorescens having none. We identified several P. aeruginosa-specific core proteins (such as CntL, CntM, PlcB, Acp1, MucE, SrfA, Tse1, Tsi2, Tse3, and EsrC) that are known to play an important role in its pathogenicity. Finally, a holin family bacteriocin and a mitomycin-like biosynthetic protein were found to be core-specific for P. cholororaphis and we hypothesize that these proteins may confer a competitive advantage against other root-colonizers.
TL;DR: The combined treatment of Ag-nanoparticles and PGPR enhanced flavonoids content and the activities of defense related enzyme PAL and the antioxidant enzymes, superoxide dismutase (SOD) and catalase(CAT) in leaves of plants over control.
Abstract: BACKGROUND The present investigation aimed to evaluate the role of Plant Growth- Promoting Rhizobacteria (PGPR) and Ag-nanoparticles on two varieties (American variety, Poinsett 76 and Desi variety, Sialkot selection) of cucumber plants. METHODS Cucumber seeds prior to sowing, were inoculated with two strains of PGPR, Pseudomonas putida (KX574857) and Pseudomonas stutzeri (KX574858) at the rate of 106 cells/ml. Agnanoparticles (5ppm) were sprayed on the plant at early vegetative phase 27 d after sowing. RESULTS The proline, sugar, protein, phenolics, flavonoids, chlorophyll and carotenoids contents of leaves of plants and the activities of Phenylalanine Ammonia-Lyase (PAL), Superoxide Dismutase (SOD) and Catalase (CAT) were determined from leaves of plants at early vegetative phase. After 3 months of seeds sowing, Ag-nanoparticles enhanced the length of root but decreased the length of shoot and fresh weight of root and shoot as compared to control whereas, the leaf protein, proline, phenolics, flavonoids, chlorophyll b, total chlorophyll, sugar and Phenylalanine Ammonia-Lyase (PAL) activity of plants were increased significantly over control. Ag-nanoparticles also suppressed the effect of PGPR for root, shoot length but augmented the protein and phenolics contents of leaves of both the varieties. CONCLUSION The combined treatment of Ag-nanoparticles and PGPR enhanced flavonoids content of leaves and the activities of PAL, SOD and CAT in leaves of plants over control. Agnanoparticles effectively increased the Phenylalanine Ammonia-Lyase (PAL), Catalase (CAT) and superoxide dismutase (SOD) activities in leaves of both the varieties. Pseudomonas putida may be used either alone or in combination with Ag-nanoparticles to enhance the antioxidant and defense enzyme activities. Hence, the plant can tolerate the diseases and stresses in a much better way with higher protein and phenolics content.
TL;DR: The present study offers a new approach for synthesizing highly dispersed and efficient nanocomposites through a rationally designed approach, wherein melamine-the precursor of g-C3N4-has been intimately mixed with the AgBr precursor, silver-tetraoctylammonium bromide.
Abstract: Synthesis of nanocomposites possessing intimately mixed components is highly challenging to bring out the best possible properties of the materials. The challenge is mainly due to the difficulties ...
TL;DR: P. fragi was positively correlated with a higher number of volatiles compared to the other strains, strengthening the hypothesis that volatile compound production is strain-dependent.
Abstract: The aim of the present study was to investigate the evolution of the volatile compounds of aerobically stored sterile pork meat as a consequence of the metabolic activities of inoculated specific spoilage microorganisms. Thus, Pseudomonas fragi, Pseudomonas putida, Lactobacillus sakei and Leuconostoc mesenteroides were inoculated in monocultures, dual cultures and a cocktail culture of all strains on sterile pork meat stored aerobically at 4 and 10 °C. Microbiological and sensory analyses, as well as pH measurements, were performed, along with headspace solid-phase microextraction gas chromatography/mass spectroscopy (headspace SPME–GC/MS) analysis. Data analytics were used to correlate the volatile compounds with the spoilage potential of each stain using multivariate data analysis. The results for the sensory discrimination showed that the volatiles that dominated in spoiled samples consisted mostly of alcohols, ketones and two esters (butyl acetate and ethyl acetate), while at fresh samples, dimethyl sulfide, furans, acetoin and ethyl lactate were detected. On the other hand, 2-butanone, diacetyl and acetaldehyde were among the volatile compounds that were mainly correlated with the inoculated meat during storage. In addition, P. fragi was positively correlated with a higher number of volatiles compared to the other strains, strengthening the hypothesis that volatile compound production is strain-dependent.