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Microbial Cell Factories Feb 2024Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too...
Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.
Topics: Pseudomonas putida; Polyhydroxyalkanoates; Polyesters; Carboxylic Acids; Oxidation-Reduction
PubMed: 38365718
DOI: 10.1186/s12934-024-02310-7 -
The ISME Journal Jan 2024The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been...
The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been published so far. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase (mSHMT) and a d-serine dehydratase that plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Conjugational plasmid transfer into Pseudomonas putida KT2440 enabled this strain to grow with TRIS and with 2-hydromethylserine, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways, and database searches suggest that the TRIS degradation pathway is now globally distributed. Overall, our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales.
Topics: Pseudomonas putida; Pseudomonas; Multigene Family; Oxidation-Reduction; Metabolic Networks and Pathways
PubMed: 38365256
DOI: 10.1093/ismejo/wrad023 -
Polymers Jan 2024Infections of agricultural crops caused by pathogen ic fungi are among the most widespread and harmful, as they not only reduce the quantity of the harvest but also...
Infections of agricultural crops caused by pathogen ic fungi are among the most widespread and harmful, as they not only reduce the quantity of the harvest but also significantly deteriorate its quality. This study aims to develop unique seed-coating formulations incorporating biopolymers (polyhydroxyalkanoate and pullulan) and beneficial microorganisms for plant protection against phytopathogens. A microbial association of biocompatible endophytic bacteria has been created, including D5, A2, B5, and D7. These strains exhibited agronomically valuable properties: synthesis of the phytohormone IAA (from 45.2 to 69.2 µg mL), antagonistic activity against and (growth inhibition zones from 1.8 to 3.0 cm), halotolerance (5-15% NaCl), and PHA production (2.77-4.54 g L). A pullulan synthesized by C7 showed a low viscosity rate (from 395 Pa·s to 598 Pa·s) depending on the concentration of polysaccharide solutions. Therefore, at 8.0%, / concentration, viscosity virtually remained unchanged with increasing shear rate, indicating that it exhibits Newtonian flow behavior. The effectiveness of various antifungal seed coating formulations has been demonstrated to enhance the tolerance of barley plants to phytopathogens.
PubMed: 38337265
DOI: 10.3390/polym16030376 -
Journal of Hazardous Materials Apr 2024Marine sediments polluted from anthropogenic activities can be major reservoirs of toxic mercury species. Some microorganisms in these environments have the capacity to...
Marine sediments polluted from anthropogenic activities can be major reservoirs of toxic mercury species. Some microorganisms in these environments have the capacity to detoxify these pollutants, by using the mer operon. In this study, we characterized microbial cultures isolated from polluted marine sediments growing under diverse environmental conditions of salinity, oxygen availability and mercury tolerance. Specific growth rates and percentage of mercury removal were measured in batch cultures for a selection of isolates. A culture affiliated with Pseudomonas putida (MERCC_1942), which contained a mer operon as well as other genes related to metal resistances, was selected as the best candidate for mercury elimination. In order to optimize mercury detoxification conditions for strain MERCC_1942 in continuous culture, three different dilution rates were tested in bioreactors until the cultures achieved steady state, and they were subsequently exposed to a mercury spike; after 24 h, strain MERCC_1942 removed up to 76% of the total mercury. Moreover, when adapted to high growth rates in bioreactors, this strain exhibited the highest specific mercury detoxification rates. Finally, an immobilization protocol using the sol-gel technology was optimized. These results highlight that some sediment bacteria show capacity to detoxify mercury and could be used for bioremediation applications.
Topics: Mercury; Bacteria; Bioreactors; Environmental Pollutants
PubMed: 38335604
DOI: 10.1016/j.jhazmat.2024.133685 -
The Journal of Biological Chemistry Feb 2024The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by β-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP...
The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by β-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP decarboxylase are the two pathways that initiate bacterial fatty acid synthesis (FAS) in Escherichia coli. In addition to these two routes, we report that Pseudomonas putida F1 β-ketoacyl-ACP synthase I (FabB), in addition to playing a key role in fatty acid elongation, also initiates FAS in vivo. We report that although two P. putida F1 fabH genes (PpfabH1 and PpfabH2) both encode functional KAS III enzymes, neither is essential for growth. PpFabH1 is a canonical KAS III similar to E. coli FabH whereas PpFabH2 catalyzes condensation of malonyl-ACP with short- and medium-chain length acyl-CoAs. Since these two KAS III enzymes are not essential for FAS in P. putida F1, we sought the P. putida initiation enzyme and unexpectedly found that it was FabB, the elongation enzyme of the oxygen-independent unsaturated fatty acid pathway. P. putida FabB decarboxylates malonyl-ACP and condenses the acetyl-ACP product with malonyl-ACP for initiation of FAS. These data show that P. putida FabB, unlike the paradigm E. coli FabB, can catalyze the initiation reaction in FAS.
Topics: 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; Acyl Carrier Protein; Escherichia coli; Fatty Acid Elongases; Fatty Acids; Glycogen Synthase; Pseudomonas putida
PubMed: 38335573
DOI: 10.1016/j.jbc.2023.105600 -
MSystems Mar 2024There is growing interest in engineering KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of relies...
UNLABELLED
There is growing interest in engineering KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules ("functional modules"), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of . Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets.
IMPORTANCE
This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.
Topics: Pseudomonas putida; Gene Regulatory Networks; Genomics
PubMed: 38323821
DOI: 10.1128/msystems.00942-23 -
Green Chemistry : An International... Feb 2024Novel synthetic strategies for the production of high-value chemicals based on the 12 principles of green chemistry are highly desired. Herein, we present a proof of...
Novel synthetic strategies for the production of high-value chemicals based on the 12 principles of green chemistry are highly desired. Herein, we present a proof of concept for two novel chemo-enzymatic one-pot cascades allowing for the production of valuable fragrance and flavor aldehydes. We utilized renewable phenylpropenes, such as eugenol from cloves or estragole from estragon, as starting materials. For the first strategy, Pd-catalyzed isomerization of the allylic double bond and subsequent enzyme-mediated (aromatic dioxygenase, ADO) alkene cleavage were performed to obtain the desired aldehydes. In the second route, the double bond was oxidized to the corresponding ketone a copper-free Wacker oxidation protocol followed by enzymatic Baeyer-Villiger oxidation (phenylacetone monooxygenase from ), esterase-mediated (esterase from , PfeI) hydrolysis and subsequent oxidation of the primary alcohol (alcohol dehydrogenase from , AlkJ) to the respective aldehyde products. Eight different phenylpropene derivatives were subjected to these reaction sequences, allowing for the synthesis of seven aldehydes in up to 55% yield after 4 reaction steps (86% for each step).
PubMed: 38323304
DOI: 10.1039/d3gc04191c -
Langmuir : the ACS Journal of Surfaces... Feb 2024Avoiding microbial contamination and biofilm formation on the surfaces of aircraft fuel tanks is a major challenge in the aviation industry. The inevitable presence of...
Avoiding microbial contamination and biofilm formation on the surfaces of aircraft fuel tanks is a major challenge in the aviation industry. The inevitable presence of water in fuel systems and nutrients provided by the fuel makes an ideal environment for bacteria, fungi, and yeast to grow. Understanding how microbes grow on different fuel tank materials is the first step to control biofilm formation in aviation fuel systems. In this study, biofilms of , a model Gram-negative bacterium previously found in aircraft fuel tanks, were characterized on aluminum 7075-T6 surfaces, which is an alloy used by the aviation industry due to favorable properties including high strength and fatigue resistance. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray (EDX) showed that extracellular polymeric substances (EPS) produced by were important components of biofilms with a likely role in biofilm stability and adhesion to the surfaces. EDX analysis showed that the proportion of phosphorus with respect to nitrogen is higher in the EPS than in the bacterial cells. Additionally, different morphologies in biofilm formation were observed in the fuel phase compared to the water phase. Micro-Fourier transform infrared spectroscopy (micro-FTIR) analysis suggested that phosphoryl and carboxyl functional groups are fundamental for the irreversible attachment between the EPS of bacteria and the aluminum surface, by the formation of hydrogen bonds and inner-sphere complexes between the macromolecules and the aluminum surface. Based on the hypothesis that nucleic acids (particularly DNA) are an important component of EPS in biofilms, the impact of degrading extracellular DNA was tested. Treatment with the enzyme DNase I affected both water and fuel phase biofilms─with the cell structure disrupted in the aqueous phase, but cells remained attached to the aluminum coupons.
PubMed: 38319653
DOI: 10.1021/acs.langmuir.3c02803 -
MBio Mar 2024
PubMed: 38315009
DOI: 10.1128/mbio.03352-23 -
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