-
Environmental Microbiology Jun 2024The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production...
The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.
Topics: Pseudomonas putida; Host Specificity; Prophages; Pseudomonas Phages; Estonia; Bacteriophages
PubMed: 38863081
DOI: 10.1111/1462-2920.16671 -
Trends in Biotechnology Jan 2024Bioelectrochemical systems (BESs), a group of promising integrated systems that combine the advantages of biotechnology and electrochemical techniques, offer new... (Review)
Review
Bioelectrochemical systems (BESs), a group of promising integrated systems that combine the advantages of biotechnology and electrochemical techniques, offer new opportunities to address environmental and energy challenges. Exoelectrogens capable of extracellular electron transfer (EET) are the critical factor enabling electrocatalytic activity in BESs. Pseudomonas putida, an aerobe widely used in environmental bioremediation, the biosynthesis of valuable chemicals, and energy bioproduction, has attracted much attention due to its unique application potential in BESs. This review provides a comprehensive understanding of the working principles, key factors, and applications of BESs using P. putida as the exoelectrogen. The challenges and perspectives for the development of BESs with P. putida as the exoelectrogen are also proposed and discussed.
PubMed: 38184440
DOI: 10.1016/j.tibtech.2023.12.002 -
Environmental Microbiology Sep 2023Pseudomonads are considered to be among the most widespread culturable bacteria in mesophilic environments. The evolutive success of Pseudomonas species can be... (Review)
Review
Pseudomonads are considered to be among the most widespread culturable bacteria in mesophilic environments. The evolutive success of Pseudomonas species can be attributed to their metabolic versatility, in combination with a set of additional functions that enhance their ability to colonize different niches. These include the production of secondary metabolites involved in iron acquisition or having a detrimental effect on potential competitors, different types of motility, and the capacity to establish and persist within biofilms. Although biofilm formation has been extensively studied using the opportunistic pathogen Pseudomonas aeruginosa as a model organism, a significant body of knowledge is also becoming available for non-pathogenic Pseudomonas. In this review, we focus on the mechanisms that allow Pseudomonas putida to colonize biotic and abiotic surfaces and adapt to sessile life, as a relevant persistence strategy in the environment. This species is of particular interest because it includes plant-beneficial strains, in which colonization of plant surfaces may be relevant, and strains used for environmental and biotechnological applications, where the design and functionality of biofilm-based bioreactors, for example, also have to take into account the efficiency of bacterial colonization of solid surfaces. This work reviews the current knowledge of mechanistic and regulatory aspects of biofilm formation by P. putida and pinpoints the prospects in this field.
Topics: Pseudomonas putida; Pseudomonas; Biofilms; Pseudomonas aeruginosa; Plants
PubMed: 37045787
DOI: 10.1111/1462-2920.16385 -
Toxics Oct 2023Heavy metals, specifically cadmium (Cd) and lead (Pb), contaminating water bodies of Madinah (Saudi Arabia), is a significant environmental concern that necessitates...
Heavy metals, specifically cadmium (Cd) and lead (Pb), contaminating water bodies of Madinah (Saudi Arabia), is a significant environmental concern that necessitates prompt action. Madinah is exposed to toxic metals from multiple sources, such as tobacco, fresh and canned foods, and industrial activities. This influx of toxic metals presents potential hazards to both human health and the surrounding environment. The aim of this study is to explore the viability of utilizing metallothionein from () as a method of bioremediation to mitigate the deleterious effects of pollution attributable to Pb and Cd. The use of various computational approaches, such as physicochemical assessments, structural modeling, molecular docking, and protein-protein interaction investigations, has enabled us to successfully identify the exceptional metal-binding properties that metallothionein displays in . The identification of specific amino acid residues, namely GLU30 and GLN21, is crucial in understanding their pivotal role in facilitating the coordination of lead and cadmium. In addition, post-translational modifications present opportunities for augmenting the capacity to bind metals, thereby creating possibilities for focused engineering. The intricate web of interactions among proteins serves to emphasize the protein's participation in essential cellular mechanisms, thereby emphasizing its potential contributions to detoxification pathways. The present study establishes a strong basis for forthcoming experimental inquiries, offering potential novel approaches in bioremediation to tackle the issue of heavy metal contamination. Metallothionein from presents a highly encouraging potential as a viable remedy for environmental remediation, as it is capable of proficiently alleviating the detrimental consequences related to heavy metal pollution.
PubMed: 37888714
DOI: 10.3390/toxics11100864 -
Nature Communications Feb 2024Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species...
Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a "sender device" in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a "receiver device" in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia.
Topics: Arabidopsis; Microbiota; Crops, Agricultural; Solanum tuberosum; Pseudomonas putida
PubMed: 38418817
DOI: 10.1038/s41467-024-45897-6 -
Chembiochem : a European Journal of... Jan 2024Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial...
Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ-Glutamyl-isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l-glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l-alaninol (2-amino-1-propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ-glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed-batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L with a product yield of 0.11 g g glycerol and a volumetric productivity of 0.24 g L h . To the best of our knowledge, this is the first fermentative production of GIPA.
Topics: Fermentation; Glycerol; Metabolic Engineering; Glutamic Acid; Propylamines
PubMed: 37987374
DOI: 10.1002/cbic.202300608 -
Applied Microbiology and Biotechnology Jun 2024Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene... (Review)
Review
Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. KEY POINTS: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways.
Topics: Ethylene Glycol; Metabolic Engineering; Metabolic Networks and Pathways; Bacteria; Pseudomonas putida; Biofuels; Escherichia coli
PubMed: 38861200
DOI: 10.1007/s00253-024-13179-2 -
Applied and Environmental Microbiology Oct 2023have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon...
have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of expression via CRISPRi led to faster growth and glucose and -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in strains, were lower under strong CCR (glucose+-coumarate) condition compared to when repression was absent (-coumarate or glucose only).IMPORTANCEA newly isolated strain, M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the gene and small RNAs in carbon catabolite repression.
Topics: Sugars; Catabolite Repression; Xylose; Pseudomonas putida; Glucose; Hexoses; Pentoses; Carbon
PubMed: 37724856
DOI: 10.1128/aem.00852-23 -
Current Opinion in Biotechnology Feb 2024The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental... (Review)
Review
The soil bacterium Pseudomonas putida, especially the KT2440 strain, is increasingly being utilized as a host for biotransformations of both industrial and environmental interest. The foundations of such performance include its robust redox metabolism, ability to tolerate a wide range of physicochemical stresses, rapid growth, versatile metabolism, nonpathogenic nature, and the availability of molecular tools for advanced genetic programming. These attributes have been leveraged for hosting engineered pathways for production of valuable chemicals or degradation/valorization of environmental pollutants. This has in turn pushed the boundaries of conventional enzymology toward previously unexplored reactions in nature. Furthermore, modifications to the physical properties of the cells have been made to enhance their catalytic performance. These advancements establish P. putida as bona fide chassis for synthetic biology, on par with more traditional metabolic engineering platforms.
Topics: Metabolic Engineering; Pseudomonas putida; Synthetic Biology; Biotransformation; Oxidation-Reduction
PubMed: 38061264
DOI: 10.1016/j.copbio.2023.103025 -
Metabolic Engineering Sep 2023Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield...
Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic C- and H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate-unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.
Topics: Pseudomonas putida; Sugars; Deuterium; Gluconates; Glucose
PubMed: 37454792
DOI: 10.1016/j.ymben.2023.07.004