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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 -
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 -
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 -
Scientific Reports Oct 2023The present study investigated the composition, abundance, and diversity of gut microbes in full-term and late-preterm infants from a medical center in eastern China. A...
The present study investigated the composition, abundance, and diversity of gut microbes in full-term and late-preterm infants from a medical center in eastern China. A total of 144 genomes of stool samples were captured for 16S rRNA metagenomic analyses. A high abundance of commensal intestinal bacteria was detected in these samples such as Phocaeicola vulgatus, Escherichia coli, and Faecalibacterium prausnitzii, indicating a relatively consistent diversity of gut microbes in the present full-term infants aged 38-40 weeks. However, late preterm infants (n = 50) with mandatory antimicrobials feeding exhibited lower diversity but a higher composition of opportunistic pathogens such as Enterococcus species. Centralized on the situation, we explored the regulatory effect of Clostridium butyricum as probiotics on these late preterm infants. The consumption of C. butyricum did not restore the composition of gut microbes altered by antimicrobials to normal levels, although several opportunistic pathogens decreased significantly after probiotic therapy including Staphylococcus aureus, Sphingomonas echinoides, and Pseudomonas putida. We also compared the effects of day-fed versus night-fed probiotics. Intriguingly, the nighttime feeding showed a higher proportion of C. butyricum compared with probiotic day-feeding. Finally, fecal metabolome and metabolites were analyzed in late preterm infants with (n = 20) or without probiotic therapy (n = 20). The KEGG enrichment analysis demonstrated that vitamin digestion and absorption, synaptic vesicle cycle, and biotin metabolism were significantly increased in the probiotic-treated group, while MSEA indicated that a series of metabolism were significantly enriched in probiotic-treated infants including glycerolipid, biotin, and lysine, indicating the complex effects of probiotic therapy on glutathione metabolism and nutrients digestion and absorption in late preterm infants. Overall, this study provided metagenomic and metabolomic profile of the gut microbes in full-term newborns and late preterm infants in eastern China. Further studies are needed to support and elucidate the role of probiotic feeding in late preterm infants with mandatory antimicrobial treatment.
Topics: Humans; Infant, Newborn; Infant; Infant, Premature; Clostridium butyricum; Gastrointestinal Microbiome; RNA, Ribosomal, 16S; Biotin; East Asian People; Probiotics
PubMed: 37907561
DOI: 10.1038/s41598-023-45586-2 -
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 -
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 -
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 -
Synthetic and Systems Biotechnology Sep 2023Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. spp. have broad...
Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. spp. have broad metabolic potential for degrading aromatic compounds. The objective of this study was to develop a "biological funneling" strategy based on genetic modification to convert complex aromatic compounds into -muconate (ccMA) using B6-2 and MPDS as biocatalysts. The engineered strains B6-2 (B6-2ΔcatBΔsalC) and MPDS (MPDSΔsalC(pUCP18k-catA)) thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA, attaining molar conversions of 95.3% (ccMA/biphenyl) and 100% (ccMA/naphthalene). Under mixed substrates, B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration. Inserting exogenous clusters like and allowed B6-2 recombinant strains to convert benzene and toluene to ccMA. In mixed substrates, constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene; the highest molar conversion rate of ccMA from mixed substrates was 85.2% when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA) incubation with biphenyl and naphthalene. This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.
PubMed: 37637202
DOI: 10.1016/j.synbio.2023.08.001 -
Scientific Reports Dec 2023Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is...
Pseudomonads are ubiquitous bacteria with importance in medicine, soil, agriculture, and biomanufacturing. We report a novel Pseudomonas putida phage, MiCath, which is the first known phage infecting P. putida S12, a strain increasingly used as a synthetic biology chassis. MiCath was isolated from garden soil under a tomato plant using P. putida S12 as a host and was also found to infect four other P. putida strains. MiCath has a ~ 61 kbp double-stranded DNA genome which encodes 97 predicted open reading frames (ORFs); functions could only be predicted for 48 ORFs using comparative genomics. Functions include structural phage proteins, other common phage proteins (e.g., terminase), a queuosine gene cassette, a cas4 exonuclease, and an endosialidase. Restriction digestion analysis suggests the queuosine gene cassette encodes a pathway capable of modification of guanine residues. When compared to other phage genomes, MiCath shares at most 74% nucleotide identity over 2% of the genome with any sequenced phage. Overall, MiCath is a novel phage with no close relatives, encoding many unique gene products.
Topics: Bacteriophages; Genome, Viral; Pseudomonas putida; DNA, Viral; Nucleoside Q; Sequence Analysis, DNA; Soil; Open Reading Frames; Phylogeny
PubMed: 38071193
DOI: 10.1038/s41598-023-48634-z -
RSC Advances Jan 2024Biocatalytic approaches are used widely for the synthesis of amines from abundant or low cost starting materials. This is a fast-developing field where novel enzymes and...
Biocatalytic approaches are used widely for the synthesis of amines from abundant or low cost starting materials. This is a fast-developing field where novel enzymes and enzyme combinations emerge quickly to enable the production of new and complex compounds. Natural multifunctional enzymes represent a part of multi-step biosynthetic pathways that ensure a one-way flux of reactants. , they confer a selective advantage increased reaction rates and chemical stability or prevention of toxicity from reactive intermediates. Here we report the identification and analysis of a natural transaminase fusion, PP_2782, from KT2440, as well as three of its thermophilic homologs from , , and . Both the fusions and their truncated transaminase-only derivatives showed good activity with unsubstituted aliphatic and aromatic aldehydes and amines, as well as with a range of α-keto acids, and l-alanine, l-glutamate, and l-glutamine. Through structural similarity, the fused domain was recognised as the acyl-[acyl-carrier-protein] reductase that affects reductive chain release. These natural transaminase fusions could have a great potential for industrial applications.
PubMed: 38298934
DOI: 10.1039/d3ra07081f