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Microorganisms Mar 2024The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The...
The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium WP1 as the feeder strain for growth and the recombinant production of PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of in cell-containing culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by KT2440.
PubMed: 38543580
DOI: 10.3390/microorganisms12030529 -
Nature Communications Mar 2024To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory...
To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.
Topics: Xylose; Pseudomonas putida; Transaldolase; Metabolic Engineering; Pentose Phosphate Pathway
PubMed: 38531855
DOI: 10.1038/s41467-024-46812-9 -
Microbial Biotechnology Mar 2024Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell...
Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Metabolic Engineering; Escherichia coli; Synthetic Biology
PubMed: 38528784
DOI: 10.1111/1751-7915.14423 -
Cell Reports Apr 2024Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable...
Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Nutrients; Carbon; Nitrogen; Polyethylene Terephthalates
PubMed: 38517887
DOI: 10.1016/j.celrep.2024.113979 -
Gene expression reprogramming of in response to arginine through the transcriptional regulator ArgR.Microbiology (Reading, England) Mar 2024Different bacteria change their life styles in response to specific amino acids. In (now ) KT2440, arginine acts both as an environmental and a metabolic indicator that...
Different bacteria change their life styles in response to specific amino acids. In (now ) KT2440, arginine acts both as an environmental and a metabolic indicator that modulates the turnover of the intracellular second messenger c-di-GMP, and expression of biofilm-related genes. The transcriptional regulator ArgR, belonging to the AraC/XylS family, is key for the physiological reprogramming in response to arginine, as it controls transport and metabolism of the amino acid. To further expand our knowledge on the roles of ArgR, a global transcriptomic analysis of KT2440 and a null mutant growing in the presence of arginine was carried out. Results indicate that this transcriptional regulator influences a variety of cellular functions beyond arginine metabolism and transport, thus widening its regulatory role. ArgR acts as positive or negative modulator of the expression of several metabolic routes and transport systems, respiratory chain and stress response elements, as well as biofilm-related functions. The partial overlap between the ArgR regulon and those corresponding to the global regulators RoxR and ANR is also discussed.
Topics: Arginine; Repressor Proteins; Pseudomonas; Gene Expression; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 38511653
DOI: 10.1099/mic.0.001449 -
Microbial Biotechnology Mar 2024Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other...
Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacI /P system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H O and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g benzaldehyde and 0.089 g 2-HPP g cell dry weight h , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.
Topics: Benzaldehydes; Pseudomonas putida; Deinococcus; Hydroxypropiophenone; Acetaldehyde
PubMed: 38498302
DOI: 10.1111/1751-7915.14448 -
Cureus Feb 2024Nasal septum perforation (NSP) occurs secondary to many underlying etiologies, including facial trauma, drug use, malignancy, infection, or autoimmune disease. We...
A Case Report of Pseudomonas Infection in a Patient With Nasal Septum Perforation, Cocaine Use Disorder, and a Perinuclear Anti-neutrophil Cytoplasmic Antibody (p-ANCA)-Positive Assay.
Nasal septum perforation (NSP) occurs secondary to many underlying etiologies, including facial trauma, drug use, malignancy, infection, or autoimmune disease. We present the case of a 39-year-old female with a past medical history of cocaine use disorder who presented with symptoms concerning facial cellulitis unresponsive to antibiotic therapy. Physical exam and subsequent imaging revealed the presence of NSP. The patient underwent a full workup exploring potential etiologies of NSP in the setting of cocaine use disorder, with lab results indicating and cellulitis as well as a positive perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA) assay. This case highlights the importance of maintaining a broad differential diagnosis for the etiology of NSP and avoiding anchoring bias.
PubMed: 38476784
DOI: 10.7759/cureus.54022 -
BMC Genomics Mar 2024In every omics experiment, genes or their products are identified for which even state of the art tools are unable to assign a function. In the biotechnology chassis...
In every omics experiment, genes or their products are identified for which even state of the art tools are unable to assign a function. In the biotechnology chassis organism Pseudomonas putida, these proteins of unknown function make up 14% of the proteome. This missing information can bias analyses since these proteins can carry out functions which impact the engineering of organisms. As a consequence of predicting protein function across all organisms, function prediction tools generally fail to use all of the types of data available for any specific organism, including protein and transcript expression information. Additionally, the release of Alphafold predictions for all Uniprot proteins provides a novel opportunity for leveraging structural information. We constructed a bespoke machine learning model to predict the function of recalcitrant proteins of unknown function in Pseudomonas putida based on these sources of data, which annotated 1079 terms to 213 proteins. Among the predicted functions supplied by the model, we found evidence for a significant overrepresentation of nitrogen metabolism and macromolecule processing proteins. These findings were corroborated by manual analyses of selected proteins which identified, among others, a functionally unannotated operon that likely encodes a branch of the shikimate pathway.
Topics: Pseudomonas putida; Proteome; Multiomics; Biotechnology; Operon
PubMed: 38468234
DOI: 10.1186/s12864-024-10082-y -
MicroLife 2024Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly...
Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of 'fungal highways' (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in -formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.
PubMed: 38463165
DOI: 10.1093/femsml/uqae004 -
Communications Biology Mar 2024Pseudomonas aeruginosa, a common nosocomial pathogen, relies on siderophores to acquire iron, crucial for its survival in various environments and during host...
Pseudomonas aeruginosa, a common nosocomial pathogen, relies on siderophores to acquire iron, crucial for its survival in various environments and during host infections. However, understanding the molecular mechanisms of siderophore regulation remains incomplete. In this study, we found that the BfmRS two-component system, previously associated with biofilm formation and quorum sensing, is essential for siderophore regulation under high osmolality stress. Activated BfmR directly bound to the promoter regions of pvd, fpv, and femARI gene clusters, thereby activating their transcription and promoting siderophore production. Subsequent proteomic and phenotypic analyses confirmed that deletion of BfmRS reduces siderophore-related proteins and impairs bacterial survival in iron-deficient conditions. Furthermore, phylogenetic analysis demonstrated the high conservation of the BfmRS system across Pseudomonas species, functional evidences also indicated that BfmR homologues from Pseudomonas putida KT2440 and Pseudomonas sp. MRSN12121 could bind to the promoter regions of key siderophore genes and osmolality-mediated increases in siderophore production were observed. This work illuminates a novel signaling pathway for siderophore regulation and enhances our understanding of siderophore-mediated bacterial interactions and community establishment.
Topics: Humans; Siderophores; Pseudomonas aeruginosa; Osmotic Pressure; Phylogeny; Proteomics; Iron; Pseudomonas; Pseudomonas Infections
PubMed: 38461208
DOI: 10.1038/s42003-024-05995-z