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Nature Communications Jan 2024Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic...
Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon-carbon (C-C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C-C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis-muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O-demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin.
PubMed: 38286984
DOI: 10.1038/s41467-024-45038-z -
Nature Communications Jun 2024Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding...
Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.
Topics: Adenosine Triphosphate; Biosensing Techniques; Escherichia coli; Pseudomonas putida; Fatty Acids; Polyhydroxyalkanoates; Energy Metabolism; Carbon; Oleic Acid
PubMed: 38906854
DOI: 10.1038/s41467-024-49579-1 -
Microbial Cell Factories Feb 2024We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a...
We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the "wholesale" method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.
Topics: Styrene; Pseudomonas; Carboxy-Lyases; Pseudomonas putida; Phenylalanine; Cinnamates
PubMed: 38419048
DOI: 10.1186/s12934-024-02341-0 -
Microorganisms Sep 2023An experimental study by the Paul-Ehrlich Institute (PEI) demonstrated that temperatures between 35 and 37 °C are too high for the growth of some bacterial strains...
An experimental study by the Paul-Ehrlich Institute (PEI) demonstrated that temperatures between 35 and 37 °C are too high for the growth of some bacterial strains (e.g., ), leading to false negative results. Thus, the question of whether it is necessary to adapt incubation temperatures for the microbiological control of blood products, especially platelet concentrates (PCs), to enhance safety and regulatory compliance has arisen. In order to further elucidate this issue, the growth capability of different bacterial strains of interest in PCs and the detection efficacy of cultivation of these at different incubation temperatures must be taken into account. Therefore, we inoculated PCs with 46 different strains (3-6 PCs from different donors per strain) from different origins (PC isolates, reference strains) and stored PCs at 20-22 °C under constant agitation. On day three of storage, the inoculated PCs were sampled; aerobic and anaerobic culture bottles (BacT/Alert AST/NST) were each inoculated with 5 mL of sample, and culture bottles were incubated at 25 and 35 °C using the automated BacT/Alert Dual-temperature system. Bacterial proliferation was enumerated using a colony-forming assay. All strains of ( = 5), spp. ( = 11), spp. ( = 5), and spp. ( = 4) and most strains (4 of 5) tested showed the capability to grow in most inoculated PCs, revealing a faster time to detection (TTD) at an incubation temperature of 35 °C. The tested ( = 3) strains showed a noticeably reduced capability to grow in PCs. Nonetheless, those with a notable growth capability revealed a faster TTD at an incubation temperature of 35 °C. Only one of the four strains tested (strain ATCC 13525) was able to grow in PCs, showing a faster TTD at an incubation temperature of 25 °C but also detection at 35 °C. The commonly detected bacteria involved in the bacterial contamination of PCs showed a superior TTD at 35 °C incubation. Only one strain showed superior growth at 25 °C; however, the microbiological control at 35 °C did not fail to identify this contamination. In conclusion, the use of PC screening using a dual-temperature setting for microbiological control is presently not justified according to the observed kinetics.
PubMed: 37764194
DOI: 10.3390/microorganisms11092350 -
Microorganisms Feb 2024A Special Issue of devoted to 'Microbial Biocatalysis and Biodegradation' would be incomplete without some form of acknowledgement of the many important roles that... (Review)
Review
A Special Issue of devoted to 'Microbial Biocatalysis and Biodegradation' would be incomplete without some form of acknowledgement of the many important roles that dioxygen-dependent enzymes (principally mono- and dioxygenases) play in relevant aspects of bio-oxygenation. This is reflected by the multiple strategic roles that dioxygen -dependent microbial enzymes play both in generating valuable synthons for chemoenzymatic synthesis and in facilitating reactions that help to drive the global geochemical carbon cycle. A useful insight into this can be gained by reviewing the evolution of the current status of 2,5-diketocamphane 1,2-monooxygenase (EC 1.14.14.108) from (+)-camphor-grown ATCC 17453, the key enzyme that promotes the initial ring cleavage of this natural bicyclic terpene. Over the last sixty years, the perceived nature of this monooxygenase has transmogrified significantly. Commencing in the 1960s, extensive initial studies consistently reported that the enzyme was a monomeric true flavoprotein dependent on both FMNH and nonheme iron as bound cofactors. However, over the last decade, all those criteria have changed absolutely, and the enzyme is currently acknowledged to be a metal ion-independent homodimeric flavin-dependent two-component mono-oxygenase deploying FMNH as a cosubstrate. That transition is a paradigm of the ever evolving nature of scientific knowledge.
PubMed: 38399793
DOI: 10.3390/microorganisms12020389 -
Plants (Basel, Switzerland) Aug 2023Wheat stripe rust, caused by f. sp. (), is a destructive disease that causes significant yield losses in wheat production worldwide, including in Egypt. The use of...
Wheat stripe rust, caused by f. sp. (), is a destructive disease that causes significant yield losses in wheat production worldwide, including in Egypt. The use of biocontrol agents is among the best eco-friendly management strategies to control this disease, as they are more sustainable and environmentally friendly than traditional chemical control methods. In a comparative analysis, antioxidant enzyme activity and various management approaches were compared with two bacterial biocontrol agents, and . This study showed the remarkable efficacy of endophytic bacteria, and , in mitigating wheat stripe rust infection across three wheat varieties, namely Misr1, Gimmeiza11, and Sids12. exhibited superior performance compared to , resulting in infection types of 1 and 2.66, respectively, following inoculation. The highest reduction rate was observed with Tilit fungicide (500 ppm), followed by B. subtilis and Salicylic acid (1000 ppm), respectively. Variations in wheat varieties' response to infection were observed, with Misr1 exhibiting the lowest infection and Sids12 showing high susceptibility. Among the tested inducers, Salicylic acid demonstrated the greatest reduction in disease infection, followed by Indole acetic acid, while Oxalic acid exhibited the lowest decrease. Additionally, the study evaluated the activities of five antioxidant enzymes, including Catalase, Ascorbate peroxidase (APX), glutathione reductase (GR), Superoxide dismutase (SOD), and peroxidase (POX), in the wheat-stripe rust interaction under different integrated management approaches. The wheat variety Misr1 treated with Tilit (500 ppm), B. subtilis, Salicylic acid, Montoro (500 ppm), and exhibited the highest increase in all enzymatic activities. These findings provide valuable insights into the effectiveness of and as biocontrol agents for wheat stripe rust control in Egypt, emphasizing their potential role in sustainable, integrated, and environmentally friendly management practices.
PubMed: 37631164
DOI: 10.3390/plants12162954 -
Nature Chemical Biology Nov 2023The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths...
The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O. The encoded mutations cluster around a putative O tunnel, increasing flexibility and accessibility to O in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.
Topics: Rats; Animals; Nicotine; Oxygen; Oxidoreductases; Oxidation-Reduction; Pseudomonas putida
PubMed: 37770699
DOI: 10.1038/s41589-023-01426-y -
Plants (Basel, Switzerland) Dec 2023Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic...
Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic stress and enhance crop productivity. However, it should be noted that rhizobacteria employ different approaches to deal with salt stress conditions and successfully colonize roots. The objective of this study was to investigate the effect of salt stress on bacterial survival mechanisms such as mobility, biofilm formation, and the autoaggregation capacity of three plant growth-promoting strains: SJ04, WCS417r, and GB03. These strains were grown in diluted LB medium supplemented with 0, 100, 200, or 300 mM NaCl. Swimming and swarming mobility were evaluated in media supplemented with 0.3 and 0.5% agar, respectively. Biofilm formation capacity was quantified using the crystal violet method, and the autoaggregation capacity was measured spectrophotometrically. In addition, we evaluated in vitro the capacity of the strains to ameliorate the effects of saline stress in . The study found that the GB03 strain exhibited enhanced swarming mobility when the salt concentration in the medium increased, resulting in a two-fold increase in the halo diameter at 300 mM. However, high concentrations of NaCl did not affect the swimming mobility. In contrast, swimming motility was reduced in WCS417r and SJ04 under salt stress. On the other hand, exposure to 300 mM NaCl resulted in a 180% increase in biofilm formation and a 30% rise in the percentage of autoaggregation in WCS417r. Conversely, the autoaggregation percentage of the strains SJ04 and GB03 remained unaffected by saline stress. However, for GB03, biofilm formation decreased by 80% at 300 mM. Simultaneously, inoculation with the three evaluated strains alleviated the detrimental effects of salinity on plant growth. Under 150 mM salt stress, all strains showed increased fresh weight, with GB03 and WCS417r improving by 40% and SJ04 exhibiting the most remarkable effect with a 70% rise compared to non-inoculated plants. Despite their different strategies for mitigating salt stress, the application of these strains presents a promising strategy for effectively mitigating the negative consequences of salt stress on plant cultivation.
PubMed: 38068694
DOI: 10.3390/plants12234059 -
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 -
Metabolic Engineering Jan 2024For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared...
For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.
Topics: Pseudomonas putida; Carbon; Metabolic Networks and Pathways; Plasmids
PubMed: 37918614
DOI: 10.1016/j.ymben.2023.10.005