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Efficient utilization of all available carbons from lignocellulosic biomass is critical for the economic efficiency of a bioconversion process to produce renewable bioproducts. This dissertation aims to understand the fundamentals of bioconversion of biomass-derived carbons to polyhydroxyalkoate (PHA) by Pseudomonas strain. The key contributions of this work include the elucidation of two distinct kinetic patterns of PHA accumulation with benzoate under nitrogen-limited and surplus conditions (ammonium chloride form) in Pseudomonas putida KT2440. Moreover, a novel strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in P. putida KT2440—was developed for the first time, which simultaneously improved both cell biomass and PHA production. NMR metabolite analysis and global proteome profiles suggested the stimulated oxidative-stress responses altered the intracellular NAD+/NADH and NADPH/NADP+ ratios, up-regulated the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis. Furthermore, the benzoate addition inhibited cell biomass and PHA production in the glucose system, presenting a divergent effect to glycerol. Global proteomics results revealed that in both glucose and glycerol co-feeding systems, the addition of benzoate dynamically regulated the energy-related pathways such as the ED/PP pathway, trehalose, and PHA biosynthesis pathways during early and late fermentation periods. Redox proteomics analysis results further confirmed that co-feeding benzoate with glucose dynamically regulates the energy generation pathway by increasing protein thiol oxidation in the PP pathway and glyoxylate shunt while decreasing fatty acid and trehalose metabolism, resulting in a smaller cell dry weight and PHA yield. These results showed that aromatics would not only serve as the carbon source but also generate distinct redox environments and finally reflected by different PHA accumulation patterns. In addition, the mechanisms of bacterial lignin depolymerization with P. putida were investigated via secretomic analysis. Depolymerization reaction mechanisms involved in a couple of oxidoreductases such as Dyp, multicopper oxidase, NAD(P)H dehydrogenase, glutathione reductase, glutathione S-transferases, choline dehydrogenase, and FAD-binding oxidoreductase, followed by β-Ο-4, β-β, β-5, and β-1 cleavage and aromatic intermediates release (e.g., vanillin and vanillic acid).

Plant Growth Promoting Rhizobacteria (PGPR) represent a microbial community that exerts growth-promoting capabilities in plants by various mechanisms. Among the PGPR genera, Pseudomonas spp. deserves special attention. It is due to its characteristic traits, like the production of phytohormones and siderophores, solubilization of minerals and phosphates, and plant protection from biotic and abiotic stress. These PGPR properties depend on the microorganism and its plant counterpart. The use of microbial strains as bioinoculants must consider the physiological and economic aspects of the process, and the plant growth stimulating effect has to be checked and proved. This study aimed to explore the PGP capacity of Pseudomonas putida 1046 strain in a model plant system of the economically important corn culture (Zea mays). The effect of the strain’s metabolic status on the plant germination capacity was evaluated. Bacterial cultures, grown 16 h and 48 h, were explored for the treatment of the corn seeds at three experimental concentrations: 0.1, 0.2, and 0.4%, and monitoring of their germination capacity through the growth indicators length of the radicle, length of the coleoptile, and the number of lateral roots. The data obtained outline the positive effect of Pseudomonas putida 1046 on the germination capacity of corn when applied at 0.2% concentration. The in vitro treatment of the model plants with 0.2% suspension resulted in a 22.87%–28.33% increase in the length of the radicle, a 35.96%–49.56% increase in the length of the coleoptile, and a 5.41–16.67% increase in the number of the lateral roots. High values of the vigour index (2125 for 16 h and 2721 for 48 h culture) were also registered. The strain’s ability to produce siderophores of hydroximate type and exhibit phosphate solubilizing activity is proved. The optimal treatment parameters of the corn seeds comprise the application of 0.2% suspension of 16 h grown Pseudomonas putida 1046 strain for five days.

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen

Objective:The antimicrobial activity was evaluated using flavonoid extract of leaf; stem; bark and fruit ofMimusops elengiL. Methodology:The flavonoids were extracted using cold extraction method. Antimicrobial activity of free and bound flavonoid extract was evaluated by disc diffusion assay against bacterial strainsviz.Escherichia coli;Bacillus subtilis;Pseudomonas putida. Minimum inhibitory concentration (MIC) of the extract was evaluated through micro broth dilution method. Results:Free flavonoid plant part extract ofM. elengiL. exhibited significant activity againstEscherichia coli;Bacillus subtilisand show no activity againstPseudomonas putida. Free flavonoid extract of bark of plant showed maximum inhibitory activity (DIZ = 14.0+1.00 mm) at 50mg/ml againstEscherichia coliwith MIC of 12.5 mg/ml. Bound flavonoid plant part extract ofM. elengiL. exhibited significant activity againstEscherichia coli;Pseudomonas putidawith no activity againstBacillus subtilis.Bound flavonoid extracts of leaf significantly showed maximum antibacterial activity (DIZ =21.0+1.00 mm) at 50mg/ml againstEscherichia coliwith MIC of 6.25 mg/ml.Bound flavonoid extracts of bark also showed maximum antibacterial activity (DIZ =19.33+1.15 mm) at 50mg/ml againstPseudomonas putidawith MIC of 6.25 mg/ml. Conclusion:Results obtained advocates that use of plant parts especially leaf and bark of the selected plant could be used as natural antimicrobial agent against tested microorganism and upcoming resistant pathogens.

Metabolic engineering of microorganisms aims to design strains capable of producing valuable compounds under relevant industrial conditions and in an economically competitive manner. From this perspective, and beyond the need for a catalyst, biomass is essentially a cost-intensive, abundant by-product of a microbial conversion. Yet, few broadly applicable strategies focus on the optimal balance between product and biomass formation. Here, we present a genetic control module that can be used to precisely modulate growth of the industrial bacterial chassis Pseudomonas putida KT2440. The strategy is based on the controllable expression of the key metabolic enzyme complex pyruvate dehydrogenase (PDH) which functions as a metabolic valve. By tuning the PDH activity, we accurately controlled biomass formation, resulting in six distinct growth rates with parallel overproduction of excess pyruvate. We deployed this strategy to identify optimal growth patterns that improved the production yield of 2-ketoisovalerate and lycopene by 2.5- and 1.38-fold, respectively. This ability to dynamically steer fluxes to balance growth and production substantially enhances the potential of this remarkable microbial chassis for a wide range of industrial applications.

Ethanol has recently been demonstrated as a suitable carbon source for acetyl-CoA-derived products with high theoretical yield. Herein, the short-chain-length polyhydroxyalkanoates production pathway was constructed in an industrial platform P. putida KT2440, allowing the engineered strain to produce 674.97 ± 22.3 mg/L of Polyhydroxybutyrate (PHB) from ethanol as sole carbon source. Furthermore, the ethanol catabolic pathway was reconstructed to enhance the acetyl-coA pool by expressing the novel Aldehyde dehydrogenases from Klebsiella pneumonia and Dickeya zeae, resulting in a titer of 1385.34 ± 16.5 mg/L and 9300 ± 0.56 mg/L of PHB in shake flask and fermenter, respectively. Furthermore, transcriptome analysis was conducted to provide insights into the central metabolic pathways and different expression patterns in response to changes in substrate. Additionally, the production of co-polymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) was shown using glycerol and ethanol as co-substrates from recombinant P. putida KT2440. This work demonstrates the potential of P. putida KT2440 as a promising industrial platform for short-chain-length PHAs production from structurally unrelated carbon sources.

International audience; The mesoporous iron polycarboxylate MIL-100(Fe) was synthesized in the presence of Pseudomonas putida bacteria. The synthesis was performed under green conditions, i.e., pure aqueous media at 30 °C that were compatible with the preservation of the cell membrane integrity. Interestingly, the resulting biohybrid exhibited a very different microstructure than a physical mixture of the two components, as it led to the formation of a novel living material featuring an exoskeleton encapsulating individual bacteria cell. Remarkably, TEM and STEM observations on cross sections revealed that this shell was not directly in contact with the cell wall, suggesting the exopolysaccharides network promotes strong interactions with the MOF precursors, leading to high proximity between the two components.

The dissemination of plasmid-borne antibiotic resistance genes (ARGs) among bacteria is becoming a global challenge to the "One Health" concept. During conjugation, the donor/recipient usually encounter diverse stresses induced by the surrounding environment. Previous studies mainly focused on the effects of oxidative stress on plasmid conjugation, but ignored the potential contribution of reductive stress (RS), the other side of the intracellular redox spectrum. Herein, we demonstrated for the first time that RS induced by dithiothreitol could significantly boost the horizontal transfer of plasmid RP4 from

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO2—but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

115 p.-4 fig.-45 fig. supl.-14 tab supl., Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of thesematerials, however, present major barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium, Pseudomonas putida, to funnel these oxygenated compounds into a single exemplary chemical product, either b-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products., Funding was provided by the US Department of Energy, Office of Energy Efficiency and Renewable Energy, Advanced Manufacturing Office (AMO), and Bioenergy Technologies Office (BETO). This work was performed as part of the BOTTLE Consortium and was supported by AMO and BETO under contract no. DE-AC36- 08GO28308 with the National Renewable Energy Laboratory (NREL),operated by the Alliance for Sustainable Energy, LLC. The BOTTLE Consortium includes members from MIT, funded under contract no. DE-AC36-08GO28308 with NREL. Contributions by S.S.S. were supported by the US Department of Energy, Office of Basic Energy Sciences, under award no. DEFG02-05ER15690.

Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their environmental niches. Here we show that the plant root colonizer Pseudomonas putida strain IsoF is able to kill a wide range of soil and plant-associated Gram-negative bacteria with the aid of a type IVB secretion system (T4BSS) that delivers a toxic effector into bacterial competitors in a contact-dependent manner. This extends the range of targets of T4BSSs—so far thought to transfer effectors only into eukaryotic cells—to prokaryotes. Bioinformatic and genetic analyses showed that this killing machine is entirely encoded by the kib gene cluster located within a rare genomic island, which was recently acquired by horizontal gene transfer. P. putida IsoF utilizes this secretion system not only as a defensive weapon to kill bacterial competitors but also as an offensive weapon to invade existing biofilms, allowing the strain to persist in its natural environment. Furthermore, we show that strain IsoF can protect tomato plants against the phytopathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF can be exploited for pest control and sustainable agriculture.

10 páginas.- 4 figuras.- 4 tablas.- 45 referencias.- The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.2c03149, Recent research has demonstrated that chemotactic bacteria can disperse inside microsized pores while traveling toward favorable conditions. Microbe–microbe cotransport might enable nonmotile bacteria to be carried with motile partners to enhance their dispersion and reduce their deposition in porous systems. The aim of this study was to demonstrate the enhancement in the dispersion of nonmotile bacteria (Mycobacterium gilvum VM552, a polycyclic aromatic hydrocarbon-degrader, and Sphingobium sp. D4, a hexachlorocyclohexane-degrader, through micrometer-sized pores near the exclusion-cell-size limit, in the presence of motile Pseudomonas putida G7 cells. For this purpose, we used bioreactors equipped with two chambers that were separated with membrane filters with 3, 5, and 12 μm pore sizes and capillary polydimethylsiloxane (PDMS) microarrays (20 μm × 35 μm × 2.2 mm). The cotransport of nonmotile bacteria occurred exclusively in the presence of a chemoattractant concentration gradient, and therefore, a directed flow of motile cells. This cotransport was more intense in the presence of larger pores (12 μm) and strong chemoeffectors (γ-aminobutyric acid). The mechanism that governed cotransport at the cell scale involved mechanical pushing and hydrodynamic interactions. Chemotaxis-mediated cotransport of bacterial degraders and its implications in pore accessibility opens new avenues for the enhancement of bacterial dispersion in porous media and the biodegradation of heterogeneously contaminated scenarios., This research was funded by European Union's Horizon 2020 research and innovation program under the Marie Sklodowska- Curie grant agreement no. 895340 (BIOTAC project) and by the Spanish Ministry of Science and Innovation (PID2019- 109700RB-C21) .

A total of 11 bacterial strains capable of completely degrading 2-butoxyethanol (2-BE) were isolated from forest soil, a biotrickling filter, a bioscrubber, and activated sludge, and identified by 16S rRNA gene sequence analysis. Eight of these strains belong to the genus Pseudomonas; the remaining three strains are Hydrogenophaga pseudoflava BOE3, Gordonia terrae BOE5, and Cupriavidus oxalaticus BOE300. In addition to 2-BE, all isolated strains were able to grow on 2-ethoxyethanol and 2-propoxyethanol, ethanol, n-hexanol, ethyl acetate, 2-butoxyacetic acid (2-BAA), glyoxylic acid, and n-butanol. Apart from the only gram-positive strain isolated, BOE5, none of the strains were able to grow on the nonpolar ethers diethyl ether, di-n-butyl ether, n-butyl vinyl ether, and dibenzyl ether, as well as on 1-butoxy-2-propanol. Strains H. pseudoflava BOE3 and two of the isolated pseudomonads, Pseudomonas putida BOE100 and P. vancouverensis BOE200, were studied in more detail. The maximum growth rates of strains BOE3, BOE100, and BOE200 at 30 °C were 0.204 h-1 at 4 mM, 0.645 h-1 at 5 mM, and 0.395 h-1 at 6 mM 2-BE, respectively. 2-BAA, n-butanol, and butanoic acid were detected as potential metabolites during the degradation of 2-BE. These findings indicate that the degradation of 2-BE by the isolated gram-negative strains proceeds via oxidation to 2-BAA with subsequent cleavage of the ether bond yielding glyoxylate and n-butanol. Since Gordonia terrae BOE5 was the only strain able to degrade nonpolar ethers like diethyl ether, the degradation pathway of 2-BE may be different for this strain., Projekt DEAL, Landesgraduiertenförderung der Universität Stuttgart

A major challenge in engineering microorganisms for the desirable product is maintaining the rational balance between cell growth and production. Quorum sensing (QS)-based dynamic regulations provide a pathway-independent genetic control technology to rebalance metabolic flux for biomass and product synthesis. However, the lack of a universal method for screening QS elements and the complex design of autoinduction circuits limit their applications in metabolic engineering. Here, we developed a universal method for simple and rapid screening and evaluating various QS systems from Gram-negative bacteria, and the largest library containing 195 combinations of receiving device/signal molecules was constructed and evaluated in

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.

In this study, different sizes of microcapsules with alginate and bentonite as natural macromolecular materials were prepared to investigate the release property of Pseudomonas putida Rs-198. The characteristics of three microcapsules were evaluated by SEM, FTIR, TG-DSC, XRD and wall thickness. The sizes of three microcapsules (MA, MB, and MC) were 1,270.50, 831.79 and 42.52 μm, respectively. First, the encapsulation efficiency of three MA, MB, and MC microcapsules were 82.20%, 90.41%, and 85.84%, respectively. Second, the contact angles of MA and MB samples were similar, while smaller microcapsules MC have higher contact angle (85.05°), indicating poor hydrophilia and decreasing the swelling degrees. Third, the release cumulant of Rs-198 and macromolecule BSA linear stage was fitted to self-established mathematic model. Results show that the microcapsule size had a considerably positive effect on release detail. The large microcapsule possessed strong leak-tightness for Rs-198 as a slow-release microbial agent. Furthermore, the porosity of microcapsules determined their swelling and release and may affect bacterial growth and survival. In conclusion, the Rs-198 microcapsule with different sizes will be pertinently selected based on the characteristics of agricultural production requirements.

Background: Polyhydroxyalkanoates (PHAs) are bacteria-synthesized biopolymers under imbalanced growth conditions. These biopolymers are acknowledged as potential biomaterials for future applications because of their characteristics of biocompatibility and biodegradability, and ability to be produced rapidly, and strong functionality of mechanical resistance. This article aims to perform microbial fermentation using the Pseudomonas putida strain to identify the quantity of biopolymers, particularly of the medium-chain-length (mcl-PHA) polyhydroxyalkanoates, based on the type and quantity of the added precursors (glucose and fatty acids). Methods: To understand the microbial interaction and the mechanism involved in PHA biosynthesis, several methods were employed and microbial biomass was obtained using the Pseudomonas putida strain capable of producing PHA. The polymer production by acetone extraction was analyzed using the Soxhlet method, while the biopolymer purification was done via the methanol-ethanol treatment, after which the biomass estimation was done through spectrophotometric analysis. This was followed by measuring the dry weight of the cells and quantification of the biopolymer produced using the gas chromatography method (GC). Results: The highest PHA yield was obtained using the octanoic (17 mL in 2000 mL medium) and hexanoic acids (14 mL in 2000 mL medium) as the precursors. As a result, the octanoic acid - octanoic acid, heptanoic acid – nonanoic acid, and octanoic acid - hexanoic acid were identified as the different precursors that supported the quantity of PHA obtained. Conclusion: Among the 4 types of structurally related substrates, the Pseudomonas putida ICCF 319 strain showed a preference for the C8 sublayer for the biosynthesis of the elastomeric PHAs composed predominantly of more C8 monomers than the C6 and C10.

A 10 week feeding trial was conducted to evaluate the potential effects of ginger powder (GP) and ginger extract nanoparticles (GNPs) on the growth parameters, digestive enzymes (lipase and amylase) activities, blood hematology, blood biochemical indices, immune indices (interleukin 10, immunoglobulin M, nitric oxide, and lysozymes), antioxidant activity, histological characteristics of kidney, spleen, liver, and intestine, and resistance to Aeromonas hydrophila or Pseudomonas putida infection in Nile tilapia, Oreochromis niloticus. Fish (n = 225, 27.01 ± 0.15 g) were stocked in 15 glass tanks (50 × 40 × 60 cm) and randomly allocated to five experimental treatments (TRTs) in triplicate (15 fish/replicate, 45 fish/TRT), consisting of five isocaloric–isonitrogenous diets. The treatments comprised the basal diet (1) without any additives (control group, CON), (2) with 0.5% GP (GP0.5), (3) with 1% GP (GP1), (4) with 0.5% GNPs (GNPs0.5), and (5) with 1% GNPs (GNPs1). Fish were manually fed to satiety three times a day (at 9 a.m., 12 p.m., and 2 p.m.). Fish were weighed at the start of the experiment, then the body weight, weight gain, feed intake, and feed conversion ratio were determined at the end of the experiment. At the end of the feeding period, 15 fish/TRT were intraperitoneally inoculated with two pathogenic bacterial strains (A. hydrophila or P. putida) in two separate challenge tests. Blood samples were collected from each TRT at two aliquots for hematological and biochemical analysis at the end of the feeding period. A significant improvement in fish growth was observed in GP and GNPs TRTs compared to the control group. There were no significant changes in the total amount of feed intake/fish in response to the experimental diets. Diets enriched with GNPs, particularly the GNPs1 TRT, resulted in a significant increase (p < 0.05) in digestive enzyme activity (lipase and amylase), serum growth hormone level, proteinogram, and immune indices (lysozyme, immunoglobulin M, interleukin 10, and nitric oxide). In addition, a significant increase in hepatic antioxidant enzymes (superoxide dismutase, reduced glutathione, and catalase) was observed in fish fed with GNPs-enriched diets. Survival percentages following bacterial challenge were higher in GNPs1, followed by GP1 and GNPs0.5 TRTs. Normal histomorphology was found in liver, kidney, and spleen tissues in all experimental TRTs. We conclude that GP and GNPs could be included in Nile tilapia diets for promoting fish growth, immunity, antioxidant status, and disease resistance without harming organ functions. In particular, the most effective treatment was GNPs1.

In this work, a new Ch2 strain was isolated from soils polluted by agrochemical production wastes. This strain has a unique ability to utilize toxic synthetic compounds such as epsilon-caprolactam (CAP) as a sole carbon and energy source and the herbicide glyphosate (GP) as a sole source of phosphorus. Analysis of the nucleotide sequence of the 16S rRNA gene of Ch2 revealed that the strain belongs to the species Pseudomonas putida. This strain grew in the mineral medium containing CAP in a concentration range of 0.5 to 5.0 g/L and utilized 6-aminohexanoic acid and adipic acid, which are the intermediate products of CAP catabolism. The ability of strain Ch2 to degrade CAP is determined by a conjugative megaplasmid that is 550 kb in size. When strain Ch2 is cultured in a mineral medium containing GP (500 mg/L), more intensive utilization of the herbicide occurs in the phase of active growth. In the phase of declining growth, there is an accumulation of aminomethylphosphonic acid, which indicates that the C-N bond is the first site cleaved during GP degradation (glyphosate oxidoreductase pathway). Culture growth in the presence of GP during the early step of its degradation is accompanied by unique substrate-dependent changes in the cytoplasm, including the formation of vesicles of cytoplasmic membrane consisting of specific electron-dense content. There is a debate about whether these membrane formations are analogous to metabolosomes, where the primary degradation of the herbicide can take place. The studied strain is notable for its ability to produce polyhydroxyalkanoates (PHAs) when grown in mineral medium containing GP. At the beginning of the stationary growth phase, it was shown that, the amount and size of PHA inclusions in the cells drastically increased; they filled almost the entire volume of cell cytoplasm. The obtained results show that the strain P. putida Ch2 can be successfully used for the PHAs’ production. Moreover, the ability of P. putida Ch2 to degrade CAP and GP determines the prospects of its application for the biological cleanup of CAP production wastes and in situ bioremediation of soil polluted with GP.

Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.

Assistive eco-physiological traits are necessary for microbes to adapt and colonize at polluted niches, enabling efficient clean-up. To demarcate species distinctiveness and eco-physiological traits of aromatic compounds metabolizing Pseudomonas sp. CSV86

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism. [Abstract copyright: © 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.]

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70 % involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against Multidrug Resistant (MDR) Pseudomonas aeruginosa. Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in Pseudomonas aeruginosa.

Simultaneous biodegradation of malodorous 1-propanethiol (PT) and dimethyl sulfide (DMS) inoculated with Pseudomonas putida S-1 and Alcaligenes sp. SY1 were investigated and interactions implicated were explored. Results showed that PT was completely degraded in 33 h, while a lag of 10 h was observed for DMS degradation alone, and the lag even extended to 81 h in the binary mixture. Mechanism analysis found that the lag was mainly attributed to the exposure of DMS degrader (Alcaligenes sp. SY1), rather than PT metabolites and PT degrader. The exposure time and PT concentration influenced the lag duration much. Citric acid could effectively reduce the lag. Pseudo first-order model was proved suitable for the description of PT degradation, revealing that PT degradation could be enhanced in presence of DMS regardless of its concentration. A modified Gompertz model, incorporated the lag phase, was developed for the description of DMS degradation in the mixture, revealing that DMS degradation depended on the initial PT concentration. When the lag was not considered, PT with low-concentration could promote DMS biodegradation, while a higher concentration (＞20 mg·L−1) cast negative effect.

Type VI secretion systems (T6SS) are specific antibacterial weapons employed by diverse bacteria to protect themselves from competitors. Pseudomonas putida KT2440 possesses a functional T6SS (K1-T6SS) and exhibits antibacterial activity towards a broad range of bacteria. Here we found that the Wsp signal transduction system regulated K1-T6SS expression via synthesizing the second messenger cyclic di-GMP (c-di-GMP), thus mediating antibacterial activity in P. putida. High-level c-di-GMP produced by Wsp system repressed the transcription of K1-T6SS genes in structural operon and vgrG1 operon. Transcriptional regulator FleQ and ATPase FleN functioned as repressors in the Wsp system-modulated K1-T6SS transcription. However, FleQ and FleN functioned as activators in biofilm formation, and Wsp system promoted biofilm formation largely in a FleQ/FleN-dependent manner. Furthermore, FleQ-FleN complex bound directly to the promoter of K1-T6SS structural operon in vitro, and c-di-GMP promoted the binding. Besides, P. putida biofilm cells showed higher c-di-GMP levels and lower antibacterial activity than planktonic cells. Overall, our findings reveal a mechanism by which Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN, and demonstrate the relationship between plankton/biofilm lifestyles and antibacterial activity in P. putida.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, β-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

Polyhydroxyalkanoates (PHAs) are emerging as alternatives to plastics by replacing fossil fuels with renewable raw substrates. Herein, we present the construction of engineered Escherichia coli strains to produce short-chain-length PHAs (scl-PHAs), including the monomers 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) produced from levulinic acid (LA). First, an E. coli strain expressing genes (lvaEDABC) from the levulinic acid metabolic pathway of Pseudomonas putida KT2440 was constructed to generate 4HV-CoA and 3HV-CoA. Second, both PhaAB enzymes from Cupriavidus necator H16 were expressed to supply 3-hydroxybutyrate (3HB)-CoA from acetyl-CoA. Finally, PHA synthase (PhaCCv) from Chromobacterium violaceum was introduced for the subsequent polymerization of these three monomers. The resulting E. coli strains produced four PHAs (w/w% of dry cell weight): 9.1 wt% P(4HV), 1.7 wt% P(3HV-co-4HV), 24.2 wt% P(3HB-co-4HV), and 35.6 wt% P(3HB-co-3HV-co-4HV).

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker, the most devastating disease of kiwifruit. The control of this disease is still challenging since it relies primarily on the use of copper-based pesticides and plant-resistance inducers. The recent restrictions in the European regulations on chemical pesticide, together with the rising interest of the public opinion on sustainable production methods, organic agriculture and environment-friendly pest and disease control, led to development and optimization of methods based on biological control agents (BCAs) in fruit production. However, the efficacy of BCAs has often resulted erratic thus limiting their adoption in commercial horticultural production. The selection of beneficial microorganisms directly from the phyllosphere of the host plant is a promising approach to overcome this limitation. This work aimed at screening the kiwifruit epiphytic microbiome from three infected orchards in Portugal to identify bacterial candidates able either to directly inhibit or to interfere with Psa growth, to be used for the biological control of Psa. Strains of Pseudomonas putida and Pseudomonas poae were found to efficiently antagonize Psa on flowers (reduction from 107to 104CFU mL-1in 24 h), and colonized all susceptible organs with high surviving rates in greenhouse conditions. BCA candidates will also be evaluated for their ability to promote plant growth or induce plant resistance. These results open new perspectives for developing commercial product based either on single bacterial strain or multiple bacteria with complementary mode of action, with high efficiency in colonizing the host plant.

Throughout history, people have used products from different microorganisms and plants for many medicinal and nutritional applications. Chilli peppers have been used for spicing up food, but also for their pharmacological properties. They contain alkaloid molecules called capsaicinoids, which have been shown to activate and desensitise the heat receptor - Transient Receptor Potential Vanilloid type 1 (TRPV1) cation channel. The TRPV1 receptor is a target for pain relief treatments for a range of health conditions, therefore capsaicinoids are important drug candidates.Capsaicinoids can be manufactured by extraction directly from the chili pepper,which typically results in relatively small amounts and therefore requires large areas of land. Furthermore, capsaicinoids yield can be affected by environmental and genotypical factors. Another way of production can be through the use of synthetic biology and metabolic engineering strategies to introduce enzymatic reactions in a model microorganism. This way, plant-derived or novel capsaicinoids with potentially improved therapeutic properties can be produced from given precursors, simple sugars or renewable raw materials.The aim of this work was to engineer baker’s yeast Saccharomyces cerevisiae for a whole-cell biocatalytic production of capsaicinoids. The structure of thesecompounds is comprised of a vanilloid moiety and a fatty acyl chain joined by anamide bond. The last step for synthesis of these compounds in the plant is through condensation of the precursors vanillylamine (derived from vanillin) and a fatty acyl-CoA forming the amide bond. Amide forming reactions are very important in chemical synthesis and research has been directed towards finding efficient enzymes to perform them. Furthermore, yeast as a model microorganism has been modified to produce vanillin from glucose; however, reductive amination of vanillin to vanillylamine, or production of capsaicinoids in yeast has not been achieved previously.This thesis addresses how capsaicinoids can be produced, the challenges for in vivo reductive amination of vanillin to vanillylamine and the identification andcharacterisation of several heterologous N-acyltransferase (NAT) and CoA-ligase(CL) amide-forming enzymes. Vanillylamine was produced from vanillin, byoverexpressing a vanillin aminotransferase. The transamination reaction wasimproved by the co-expression of an alanine dehydrogenase, which also removed the need for amine donor supplementation. Cultivation conditions were shown to have an effect on the transamination, with anaerobic conditions and ethanol as a cosubstrate leading to decrease in by-product formation and improved reductive amination of vanillin to vanillylamine. Moreover, combinations of NAT and CL enzymes were evaluated for production of nonivamide (a model capsaicinoid) from vanillylamine and nonanoic acid. Finally, nonivamide, was successfully produced in vivo from precursors vanillin and nonanoic acid, after implementing strain engineering strategies and evaluating reaction conditions. Even though the NAT and CL enzyme cascade effectively produced nonivamide, the titres and yields of the process can be further improved, indicating that the amidation step is the current bottleneck in the production and requires further optimisation.Lastly, the potential of S. cerevisiae as a biosensor was evaluated by overexpressing the TRPV1 receptor and modulating its activation. This was done as a first step towards the goal of using yeast as a screening platform for strains producing capsaicinoids with activity on the TRPV1 receptor.Transferring the capsaicinoid enzyme cascade in a cell host is not straightforward and requires optimal expression of several heterologous enzymes, adjustments of reaction conditions and strategies in reducing inhibitory effects and by-products. However, biosynthesis in whole-cells may offer advantages due to simple cell derived generation of enzymes, intermediates and co-factors, positively affecting the cost and performing the reaction in aqueous environment as a more environmentally sustainable way of production.

One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module from formate to methanol using both NAD and PQQ – dependent methanol dehydrogenases. Finally, we demonstrated CO2-dependent growth through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD+ ratio. This research paves the way to engineer P. putida towards growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

Current in the milliampere range can be used for electrochemical inactivation of bacteria. Yet, bacteria-including antibiotic resistant bacteria (ARB)-may be subjected to sub-lethal conditions due to imperfect mixing or energy savings measures during electrochemical disinfection. It is not known whether such sublethal current intensities have the potential to stimulate plasmid transfer from ARB. In this study, conjugal transfer of plasmid pKJK5 was investigated between Pseudomonas putida strains under conditions reflecting electrochemical disinfection. Although the abundance of culturable and membrane-intact donor and recipient cells decreased with applied current (0–60 mA), both transconjugant density and transconjugant frequency increased. Both active chlorine and superoxide radicals were generated electrolytically, and ROS generation was induced. In addition, we detected significant over expression of a core oxidative stress defense gene (ahpCF) with current. Expression of selected conjugation related genes (traE, traI, trbJ, and trbL) also significantly correlated with current intensity. A link between ROS accumulation, SOS response and enhanced conjugation is therefore the plausible consequence of sublethal current exposure. These findings suggest that sub-lethal intensities of current can enhance conjugal plasmid transfer, and that it is essential that conditions of electrochemical disinfection (applied voltage, current density, time and mixing) be carefully controlled to avoid conjugal ARG transmission.

Deep mutational scanning is a method for protein engineering. Here, we applied it to alter the ligand specificity of the transcriptional regulator XylS from

Excessive use of chemical fertilizers, in agriculture, has negative impacts on water, soil and affects the environment and health. In recent decades, researchers have been interested in the natural benefits of natural microorganisms and how they could be a good alterna- tive to the use of chemical fertilizers. The aim of this study was to investigate the effect of soil inoculation with strains of mycorrhizae and beneficial bacteria on soil properties and productivity of table grapes. Field trials were conducted on a commercial table grape pro- duction farm (Vitis vinifiera cv. Mousca), located in northeastern Morocco. Twelve-year- old plants were used. Control plants were not inoculated (T1). The prototype plants were inoculated with 1.2 × 104 of Glomus iranicum var. tenuihypharum/100 g (T2), a mixture of 1/2 concentration of Glomus iranicum var. tenuihypharum and 1/2 concentration of Pseu- domonas putida (T3) and 1 × 108 CFU ∙ g–1 of Pseudomonas putida (T4). The inoculations were realized twice; the first inoculation was completed on July 19, 2019 while the second inoculation on February 21, 2020. Soil analyses were carried out, both physicochemical (pH, electrical conductivity (EC), salinity, % of dry matter) and microbiological proper- ties (total flora, fungi and actinobacteria). Plant growth (length of the plant, number and diameter of sticks, number of clusters per tree, number of nodes per stick, distance between nodes and bud burst), yield and fruit quality (number of berries per cluster, cluster weight, cluster length and width, pH, Brix degrees, acidity, EC and % dry matter) were measured. Results showed slight trends regarding the effects of treatments on the physicochemical and microbiological properties of the soil, plant growth and fruit quality. The number of clusters was significantly higher in Glomus (T2) Pseudomonas (T4) and Glomus than in control treatments.

Water deficit has devastating impacts on legume production, particularly with the current abrupt climate changes in arid environments. The application of plant growth-promoting rhizobacteria (PGPR) is an effective approach for producing natural nitrogen and attenuating the detrimental effects of drought stress. This study investigated the influence of inoculation with the PGPR Rhizobium leguminosarum biovar viciae (USDA 2435) and Pseudomonas putida (RA MTCC5279) solely or in combination on the physio-biochemical and agronomic traits of five diverse Vicia faba cultivars under well-watered (100% crop evapotranspiration [ETc]), moderate drought (75% ETc), and severe drought (50% ETc) conditions in newly reclaimed poor-fertility sandy soil. Drought stress substantially reduced the expression of photosynthetic pigments and water relation parameters. In contrast, antioxidant enzyme activities and osmoprotectants were considerably increased in plants under drought stress compared with those in well-watered plants. These adverse effects of drought stress reduced crop water productivity (CWP) and seed yield‐related traits. However, the application of PGPR, particularly a consortium of both strains, improved these parameters and increased seed yield and CWP. The evaluated cultivars displayed varied tolerance to drought stress: Giza-843 and Giza-716 had the highest tolerance under well-watered and moderate drought conditions, whereas Giza-843 and Sakha-4 were more tolerant under severe drought conditions. Thus, co-inoculation of drought-tolerant cultivars with R. leguminosarum and P. putida enhanced their tolerance and increased their yield and CWP under water-deficit stress conditions. This study showed for the first time that the combined use of R. leguminosarum and P. putida is a promising and ecofriendly strategy for increasing drought tolerance in legume crops.

Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by Pseudomonas strains. Phenotypic and full genome analysis revealed Pseudomonas putida PCL1760 controlling tomato foot and root rot (TFRR) solely through CNN mechanism. Although the availability of nutrients and motility are the known conditions for CNN, persistence of bacteria through dormancy by ribosomal hibernation is a key phenomenon to evade both biotic and abiotic stress. To confirm this hypothesis, rsfS gene knockout mutant of PCL1760 (SB9) was first obtained through genetic constructions and compared with the wild type PCL1760. Primarily, relative expression of rsfS in PCL1760 was conducted on tomato seedlings which showed a higher expression at the apical part (1.02 ± 0.18) of the plant roots than the basal (0.41 ± 0.13). The growth curve and persistence in ceftriaxone after the induction of starvation with rifampicin were performed on both strains. Colonization on the tomato root by CFU and qPCR, including biocontrol ability against Fusarium, was also tested. The growth dynamics of both PCL1760 and SB9 in basal and rich medium statistically did not differ (p ≤ 0.05). There was a significant difference observed in persistence showing PCL1760 to be more persistent than its mutant SB9, while SB9 (pJeM2:rsfS) was 221.07 folds more than PCL1760. In colonization and biocontrol ability tests, PCL1760 was dominant over SB9 colonizing and controlling TFRR (in total, 3.044 × 104 to 6.95 × 103 fg/µL and 55.28% to 30.24%, respectively). The deletion of the rsfS gene in PCL1760 reduced the persistence and effectiveness of the strain, suggesting persistence as one important characteristic of the CNN.

Pseudomonas bharatica CSV86 T metabolizes glucose exclusively via the intracellular phosphorylative Entner-Doudoroff pathway leading the entire glucose flux through Zwf as the strain lacks oxidative routes. This may lead to limiting the concentration of downstream metabolic intermediates.

Communications biology 5, 1363 (2022). doi:10.1038/s42003-022-04336-2, Published by Springer Nature, London

Background Bisphenol A (BPA) is a rapid spreading organic pollutant that widely used in many industries especially as a plasticizer in polycarbonate plastic and epoxy resins. BPA reported as a prominent endocrine disruptor compound that possesses estrogenic activity and fulminant toxicity. Pseudomonas putida YC-AE1 was isolated in our previous study and exerted a strong degradation capacity toward BPA at high concentrations; however, the molecular degradation mechanism is still enigmatic. Results We employed RNA sequencing to analyze the differentially expressed genes (DEGs) in the YC-AE1 strain upon BPA induction. Out of 1229 differentially expressed genes, 725 genes were positively regulated, and 504 genes were down-regulated. The pathways of microbial metabolism in diverse environments were significantly enriched among DEGs based on KEGG enrichment analysis. qRT-PCR confirm the involvement of BPA degradation relevant genes in accordance with RNA Seq data. The degradation pathway of BPA in YC-AE1 was proposed with specific enzymes and encoded genes. The role of cytochrome P450 (CYP450) in BPA degradation was further verified. Sever decrease in BPA degradation was recorded by YC-AE1 in the presence of CYP450 inhibitor. Subsequently, CYP450bisdB deficient YC-AE1 strain △ bisdB lost its ability toward BPA transformation comparing with the wild type. Furthermore, Transformation of E. coli with pET-32a-bisdAB empowers it to degrade 66 mg l−1 of BPA after 24 h. Altogether, the results showed the role of CYP450 in biodegradation of BPA by YC-AE1. Conclusion In this study we propose the molecular basis and the potential role of YC-AE1cytochrome P450 monooxygenase in BPA catabolism.

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable "Catch-22" situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established

Acetone released into the atmosphere can adversely affect human health and the environment. The aim of this work was to evaluate the performance of a laboratory-scale biotrickling filter (BTF) with bioball packing material to remove acetone vapor from contaminated air. The acetone removal efficiency was investigated in two different scenarios: with and without the inoculation of microorganisms. Three strains of bacteria, Pseudomonas putida, Rhodococcus aerolatus, and Aquaspirillum annulus, were used in the BTF. In both cases, the filter units were simultaneously operated for 100 days under three different inlet acetone concentrations (0.18 ± 0.01 g/m3, 0.25 ± 0.01 g/m3, and 0.40 ± 0.02 g/m3) and two different gas flow rates (2.54 and 5.09 m3/h). The results showed that acetone removal was greater in the filter with the inoculated bacteria. In the filter operated without inoculum, the acetone removal efficiency gradually decreased with filtration time from 90.1% to 6.1%. While employing three types of bacteria in the BTF, the efficiency of acetone removal remained relatively stable and varied between 70.2% and 97.6%. The study also revealed that bioballs can be successfully used as a packing material in air biofiltration systems designed for acetone removal from the air.

Background Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. Results P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. Conclusion Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts., Microbial Cell Factories, 21 (1), ISSN:1475-2859

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose–response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this work Pseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10–20 mGy d−1) of 239Pu and 55Fe, both alone and in combination, for a period of 20 days, and the accumulation of 239Pu and 55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with 239Pu and stable Fe (primarily 56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that 239Pu inhibited the uptake of 55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by 239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

Background Lignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production of a range of biochemicals, and pretreatment is generally regarded as indispensable for its biorefinery. However, various inhibitors that severely hinder the growth and fermentation of microorganisms are inevitably produced during the pretreatment of lignocellulose. Presently, there are few reports on a single microorganism that can detoxify or tolerate toxic mixtures of pretreated lignocellulose hydrolysate while effectively transforming sugar components into valuable compounds. Alternatively, microbial coculture provides a simpler and more efficacious way to realize this goal by distributing metabolic functions among different specialized strains. Results In this study, a novel synthetic microbial consortium, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from highly toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of the sugar metabolism pathway was unable to consume the major fermentable sugars of lignocellulosic hydrolysate but exhibited great tolerance to 10 g/L sodium acetate, 5 g/L levulinic acid, 10 mM furfural and HMF as well as 2 g/L monophenol compound. In addition, the engineered strain rapidly removed diverse inhibitors of real hydrolysate. The degradation rate of organic acids (acetate, levulinic acid) and the conversion rate of furan aldehyde were both 100%, and the removal rate of most monoaromatic compounds remained at approximately 90%. With detoxification using engineered P. putida for 24 h, the 30% (v/v) hydrolysate was fermented to 35.8 g/L lactic acid by B. coagulans with a lactic acid yield of 0.8 g/g total sugars. Compared with that of the single culture of B. coagulans without lactic acid production, the fermentation performance of microbial coculture was significantly improved. Conclusions The microbial coculture system constructed in this study demonstrated the strong potential of the process for the biosynthesis of valuable products from lignocellulosic hydrolysates containing high concentrations of complex inhibitors by specifically recruiting consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for the bioconversion of lignocellulosic biomass to other value-added biochemicals.

Summary 5‐Ketofructose (5‐KF) is a promising low‐calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane‐bound three‐subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5‐KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5‐KF. In a batch fermentation, 129 g l−1 5‐KF were formed from 150 g l−1 fructose within 23 h, corresponding to a space‐time yield of 5.6 g l−1 h−1. Besides fructose, also sucrose could be used as substrate for 5‐KF production by plasmid‐based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5‐KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5‐KF production., 5‐Ketofructose is a promising candidate to serve as a non‐nutritive sweetener. It is formed by periplasmic oxidation of fructose with a membrane‐associated fructose dehydrogenase FdhSCL from Gluconobacter japonicus that involves an FAD‐containing subunit and a trihaem cytochrome c. In this study, we demonstrate that Pseudomonas putida KT2440 can functionally express the fdhSCL genes and serve as an efficient producer of 5‐ketofructose from fructose, but also from sucrose, which is split by a periplasmic invertase also derived from G. japonicus.

Summary Whole‐cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco‐efficient route to produce phenolic amines. Here, a novel cell growth‐based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp‐SpuC‐II and ATA from Chromobacterium violaceum (Cv‐ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp‐SpuC‐II and Cv‐ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94‐ and 92‐fold increased specific transaminase activity, respectively. Whole‐cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp‐SpuC‐II and Cv‐ATA, respectively. Still, amine production was limited by a substantial re‐assimilation of the product and formation of the by‐products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv‐ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re‐assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement., In this study, Pseudomonas putida was engineered for whole‐cell conversion of ferulic acid and vanillin to vanillylamine with ammonium as nitrogen source. Furthermore, a novel growth‐based screening method to select for recombinant vanillin transaminases was developed and used to identify SpuC‐II and Cv‐ATA as suitable enzymes for in vivo biocatalysis with P. putida.

The phytoremediation procedure was conducted by Lemna gibba (L) and Typha angustifolia (T) and the bioaugmentation procedure used P. putida HM627618. The ability of the selected P. putida HM627618 to tolerate and remove PCP (200 mg L−1) was measured by high performance liquid chromatography analysis and optical density at 600 nm. Five different experiments were conducted in secondary treated wastewater for PCP testing removal (100 mg L−1) including two phytoremediation assays (T + PCP; L + PCP), three bioaugmentation-phytoremediation assays (T + B + PCP; L + B + PCP; L + T + B + PCP) and a negative control assay with PCP. Various analytical parameters were determined in this study such as bacterial count, chlorophylls a and b, COD, pH and PCP content. The main results showed that the average PCP removal by P. putida HM627618 was around 87.5% after 7 days of incubation, and 88% of PCP removal was achieved by treatment (T + B) after 9 days. During these experiments, pH, COD and chloride content showed a net increase in all treatments. The chlorophylls a and b in case of (T) and (L) Chlorophylls a and b for T and L phytoremediation showed a decrease with a value less than 10 μg/mg of fresh material after 20 days of cultivation. HIGHLIGHT The use of macrophytes with two categories: aquatic fixed and floating models to treat polychlorinated pollutants from wastewater. This study evaluated the efficiency of Lemna gibba and Typha angustifolia to remove pentachlorophenol from aqueous solution further, and the combined process of phytoremediation and bioaugmentation efficacy in PCP removal by the plant.

In this study, the optimisation of a process for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) by Pseudomonas putida KT2440 when fed with a polyethene (PE)-derived fatty acid mixture was investigated. The PE was pyrolysed to produce a hydrocarbon wax that was subsequently oxidised to produce a mixture of fatty acids, purified, and used as a PHA substrate for the growth and selection of microorganisms. Based on the shaken flask screening, a production strain, i.e., Pseudomonas putida KT2440, was selected for conducting bioreactor studies. Feeding PE-derived fatty acids in a 20-L setup resulted in high mcl-PHA yields (83.0 g L−1 CDW with 65% PHA in 25 h). Furthermore, life-cycle assessment (LCA) was conducted to determine the environmental advantages of the proposed process and its impacts compared to those of other technologies for treating PE-derived waste streams. We conclude that processing waste PE into PHA, rather than incineration, produces biodegradable material while also reducing the additional emissions that arise from traditional PE waste treatment processes, such as incineration to gain energy.

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (P lac , P tac , P tetR/tetA and P bad ) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype., To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose.