The role of fatty acid biosynthesis and degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in Pseudomonas putida

Abstract The relationship between fatty acid metabolism and PHA biosynthesis in P. putida is described. Detailed ¹H and ¹³C NMR studies were performed to investigate the structures of poly(3-hydroxyalkanoates) (PHAs) formed from carbohydrates and fatty acids. On the basis of these results, it is proposed that during growth on glucose the 3-hydroxyacyl-acyl carrier protein intermediates of the de novo fatty acid biosynthetic pathway are diverted to PHA biosynthesis. Similarly, further evidence is presented that during cultivation on fatty acids, intermediates of the β-oxidation cycle serve as precursors of PHA biosynthesis.     

A common pathway of sulfide oxidation by sulfate-reducing bacteria

Abstract Pseudomonas putida strain DMB capable of growing on 3,4-dimethylbenzoic acid as the only C and energy source was isolated by enrichment techniques. It does not utilize for growth or cooxidize the other dimethylbenzoate isomers tested. 3,4-Dimethylsalicylic acid, 3,4-dimethylphenol and 3,4-dimethylcatechol were isolated and identified by nuclear magnetic resonance and mass spectra in the reaction mixture of P. putida washed cells. The detection of the two first metabolites suggests that the initial step in the degradation of 3,4-dimethylbenzoic acid is the formation of 3,4-dimethylcyclohexa-3,5-diene-1, 2-diol-1-carboxylic acid which underwent an acid-catalyzed dehydration yielding 3,4-dimethylsalicylic acid and 3,4-dimethylphenol. Further degradation proceeds through 3,4-dimethylcatechol via the meta pathway.     

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.     

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

Growing environmental concern sparks renewed interest in the sustainable production of (bio)materials that can replace oil-derived goods. Polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in the central metabolism of producer bacteria, as they act as dynamic reservoirs of carbon and reducing equivalents. PHAs continue to attract industrial attention as a starting point toward renewable, biodegradable, biocompatible, and versatile thermoplastic and elastomeric materials. Pseudomonas species have been known for long as efficient biopolymer producers, especially for medium-chain-length PHAs. The surge of synthetic biology and metabolic engineering approaches in recent years offers the possibility of exploiting the untapped potential of Pseudomonas cell factories for the production of tailored PHAs. In this article, an overview of the metabolic and regulatory circuits that rule PHA accumulation in Pseudomonas putida is provided, and approaches leading to the biosynthesis of novel polymers (e.g., PHAs including nonbiological chemical elements in their structures) are discussed. The potential of novel PHAs to disrupt existing and future market segments is closer to realization than ever before. The review is concluded by pinpointing challenges that currently hinder the wide adoption of bio-based PHAs, and strategies toward programmable polymer biosynthesis from alternative substrates in engineered P. putida strains are proposed.     

A novel membrane stirrer system enables foam-free biosurfactant production

Bioreactors are the operative backbone, for example, for the production of biopharmaceuticals, biomaterials in tissue engineering, and sustainable substitutes for chemicals. Still, the Achilles' heel of bioreactors nowadays is the aeration which is based on intense stirring and gas sparging, yielding inherent drawbacks such as shear stress, foaming, and sterility concerns. We present the synergistic combination of simulations and experiments toward a membrane stirrer for the efficient bubble-free aeration of bioreactors. A digital twin of the bioreactor with an integrated membrane-module stirrer (MemStir) was developed with computational fluid dynamics (CFD) studies addressing the determination of fluid mixing, shear rates, and local oxygen concentration. Usability of the MemStir is shown in a foam-free recombinant production process of biosurfactants (rhamnolipids) from glucose with different strains of Pseudomonas putida KT2440 in a 3-L vessel and benchmarked against a regular aerated process. The MemStir delivered a maximal oxygen transfer rate (OTR_max) of 175 mmol L⁻¹ h⁻¹ in completely foam-free cultivations. With a high space-time yield (STY) of 118 mg_RL L⁻¹ h⁻¹ during a fed-batch fermentation, the effectiveness of the novel MemStir is demonstrated. Simulations show the generic value of the MemStir beyond biosurfactant production, for example, for animal cell cultivation.     

The Application of Adsorption Modeling and Fourier Transform Infrared Spectroscopy to the Comparison of Two Species of Plant Growth–Promoting Rhizobacteria as Biosorbents of Cadmium in Different pH Solutions

Batch experiments were performed to determine the cadmium absorption capacity of two plant growth–promoting rhizobacteria at different pH levels and in different cadmium concentrations. Comparison of the mean metal removal from two species of bacteria studied showed that Pseudomonas florescence is the superior species for removing cadmium at all cadmium concentrations. The maximum cadmium absorption by P. florescence and P. putida were at 5 mg/L of cadmium concentration in pH 6 and 7, respectively. The applicability of the Langmuir and Freundlich isotherm models was surveyed. Comparison of two isotherm parameters (Q _m and a) further confirmed that P. fluorescence was better at binding cadmium ions (52.6 and 7.7 mg/g, respectively). Adsorption reaction also was considered by Fourier transform infrared (FTIR) spectroscopy. The FTIR analysis implied that the principal functional sites in the bacterial cell walls were phosphoryl and hydroxyl, carboxyl, amide I, amide II, and amine groups.     

Transformation of 2-chloroquinoline to 2-chloro-cis-7,8-dihydro-7,8-dihydroxyquinoline by quinoline-grown resting cells of Pseudomonas putida 86

Abstract Resting cells of Pseudononas putida strain 86 were grown on quinoline transformed 2-chloroquinoline to 2-chloro- cis -7,8-dihydro-7,8-dihydroxyquinoline which was not converted further. 7,8-Dioxygenating activity was present when the enzymes of quinoline catabolism were induced. Quinoline-grown cells of strain 86 treated simultaneously with 2-chloroquinoline and D-(-)- threo -chloramphenicol to prevent protein biosynthesis also formed the cis -7,8-dihydrodiol of 2-chloroquinoline. Succinate-grown resting cells did not oxidize 2-chloroquinoline. Acid-catalyzed decomposition of 2-chloro- cis -7,8-dihydro-7,8-dihydroxyquinoline predominantly yielded 2-chloro-8-hydroxyquinoline. By analogy, accumulation of the putative dead-end metabolite 1 H -8-hydroxy-2-oxoquinoline during growth of P. putida 86 on quinoline is suggested to likewise result from dehydration of the 7,8-dihydrodiol of 1 H -2-oxoquinoline.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.     

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers. Received 15 November 1996/ Accepted in revised form 09 March 1997