This study investigates the synthesis of aromatic nitriles using an evolved variant of OxdF1 (L318F/F306Y), an aldoxime dehydratase from Pseudomonas putida F1, engineered for improved catalytic efficiency toward benzaldehyde oxime. The double OxdF1 (L318F/F306Y) mutant effectively catalyzes the conversion of various benzaldoxime derivatives to the corresponding nitriles. Due to the enzyme's inherent instability, immobilized whole-cell systems are employed in a flow reactor to improve its stability and broaden its applicability, with the biotransformation of benzaldehyde oxime and 2,6-difluorobenzaldehyde oxime serving as case studies. The enzyme's stability is markedly improved, maintaining 87% yield even after 8 h of processing in the preparation of benzonitrile. Preparation of 2,6-difluorobenzontirile poses additional challenges due to the low water solubility of both the substrate, and even more so, the product, an important intermediate in various chemical applications. To overcome solubility limitations, a segmented liquid-liquid flow system (water/cyclohexane) was implemented, significantly improving the enzyme stability. The process was run continuously for 12 h, with a conversion of ≈70% by the end of the operation. Furthermore, 2,6-difluorobenzonitrile is selectively extracted in-line using a liquid-liquid extractor, thus, facilitating its efficient recovery and purification.

Structural inferences into the catalytic behavior of a cis/trans fatty acid isomerase from Pseudomonas putida F1.

A pilot-scale treatment system was developed to manage winery wastewater (WWW) generated by small and medium wineries. The system incorporated three stages: pre-treatment for suspended solids removal and a two-step aerobic biotreatment. The biotreatment phase utilized a bioaugmented bioreactor with encapsulated Pseudomonas putida F1, employing the Small Bioreactor Platform (SBP) technology. This innovative encapsulation method enhanced the breakdown of recalcitrant compounds and accelerated the biodegradation process. The second reactor was operated as a Sequence Batch Bioreactor (SBR) to remove the remaining organics and solids. Over the 100 days of operation, the mean WWW flow rate was 0.5 m3/d with average organic loads of 3950 mg/L COD (chemical oxygen demand) and 2220 mg/L BOD (biological oxygen demand), operating with a hydraulic retention time (HRT) of 4 days. Reductions of up to 96% in BOD and 90% in COD values were observed with stable removal rates over time. The novelty of this study is that it offers a new, effective aerobic biological treatment process, embracing bioaugmentation of encapsulated biomass followed by SBR for WWW with a relatively short HRT, high organics removal, and a stable treatment process. The effluent quality from this treatment system met the regulatory requirements for release to a municipal wastewater treatment plant and potentially also for irrigation.

Pseudomonas bacteria demonstrate a great deal of metabolic diversity and consequently colonize a wide range of ecological niches. A characteristic of these bacteria is a pair of genes in tandem annotated as acyl-CoA ligases involved in fatty acid degradation. The Pseudomonas putida F1 genome is annotated as having at least nine genes encoding acyl-CoA ligases which are scattered around the chromosome excepting the tandem pair. Since similar tandem pairs are found in other pseudomonads, we have constructed and characterized deletion mutants of the tandem ligases. We report that the encoded proteins are authentic acyl-CoA ligases involved in fatty acid degradation.

Diversity in fatty acid elongation enzymes: The FabB long-chain β-ketoacyl-ACP synthase I initiates fatty acid synthesis in Pseudomonas putida F1

Engineered aldoxime dehydratase to enable the chemoenzymatic conversion of benzyl amines to aromatic nitriles

Herein we report the use of Pseudomonas putida F1 biofilms grown on carbonized cellulosic fibers to achieve biodegradation of airborne volatile organic compounds (VOCs) in the absence of any bulk aqueous-phase media. It is believed that direct exposure of gaseous VOC substrates to biomass may eliminate aqueous-phase mass transfer resistance and facilitate VOC capture and degradation. When tested with toluene vapor as a model VOC, the supported biofilm could grow optimally at 300 p.p.m. toluene and 80% relative humidity, with a specific growth rate of 0.425 day-1 . During long-term VOC biodegradation tests in a tubular packed bed reactor, biofilms achieved a toluene degradation rate of 2.5 mg gDCW -1 h-1 during the initial growth phase. Interestingly, the P. putida F1 film kept biodegrading activity even at the stationary nongrowth phase. The supported biofilms with a biomass loading of 20% (wt) could degrade toluene at a rate of 1.9 mg gDCW -1 h-1 during the stationary phase, releasing CO2 at a rate of 6.4 mg gDCW -1 h-1 at the same time (indicating 100% conversion of substrate carbon to CO2 ). All of these observations promised a new type of "dry" biofilm reactors for efficient degradation of toxic VOCs without involving a large amount of water.

Artificial neural network modeling on trichloroethylene biodegradation in a packed-bed biofilm reactor and its comparison with response surface modeling approach

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.

Simulating degradation of organic compounds accounting for the growth of microorganisms (Monod kinetics) in a fully Lagrangian framework

Even allocation of benefits stabilizes microbial community engaged in metabolic division of labor.

Phenolic compounds have been enlisted by the United States Environmental Protection Agency (USEPA) and the European Union (EU) as pollutants of priority concern. The biphenyl degradation pathway plays an essential role in prokaryote polychlorinated biphenyls degradation. Our understanding of prokaryotic pathways and their evolution has dramatically increased in recent years with the advancements in prokaryotic genome sequencing and analysis tools. In this work, we applied bioinformatics tools to study the evolution of the biphenyl degradation pathway focusing on the phylogeny and initiation of four representative species (Burkholderia xenovorans LB400, Polaromonas naphthalenivorans CJ2, Pseudomonas putida F1 and Rhodococcus jostii RHA1). These species contained partial or full concatenated genes from bph gene cluster (i.e. bphRbphA1A2A3A4BCKHJID). The aim was to establish this pathway's origin and development mode in the prokaryotic world. Genomic screening revealed that many bacterial species possess genes for the biphenyl degradation pathway. However, the micro-synteny conservation analysis indicated that massive gene recruitment events might have occurred during the evolution of the biphenyl degradation pathway. Combining with the phylogenetic positions, this work points to the evolutionary process of acquiring the biphenyl degradation pathway by different fragments through horizontal gene transfer in these bacterial groups. This study reports the first-ever evidence of the birth of this pathway in the represented species.

ABSTRACTPerfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment.

Cyanide-free synthesis of aromatic nitriles from aldoximes: Discovery and application of a novel heme-containing aldoxime dehydratase

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacyl-ACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida ΔfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the ΔfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida β-oxidation pathway gave increased accumulation. The ΔfabB ΔfabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the ΔfabF2 ΔPput_2425 double-mutant strain still accumulated suppressors at low UFA concentrations.

Abstract Toluene dioxygenase (TDO) from Pseudomonas putida F1 was engineered towards the oxyfunctionalization of bicyclic substrates. Single and double mutant libraries addressing 27 different positions, located at the active site and entrance channel were generated. In total, 176 different variants were tested employing the substrates naphthalene, 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine. Introduced mutations in positions M220, A223 and F366, exhibited major influences in terms of product formation, chemo‐, regio‐ and enantioselectivity. By semi‐rational evolution, we lighted up the TDO capability to convert bulkier substrates than its natural substrate, at unprecedented reported conversions. Thus, the most active TDO variants were applied to biocatalytic oxyfunctionalizations of 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine, enabling the production of substantial amounts of (+)‐(R)‐1,2,3,4‐tetrahydroquinoline‐4‐ol (71% isolated yield, 94% ee) and (+)‐(1S,2R)‐3‐(pyridin‐2‐yl)cyclohexa‐3,5‐diene‐1,2‐diol (60% isolated yield, 98% ee), respectively. Here, we provide a set of novel TDO‐based biocatalysts useful for the preparation of oxyfunctionalized bicyclic scaffolds, which are valuable to perform downstream synthetic processes.magnified image

Abstract Reactor design and feeding strategies are the key to successful trichloroethylene (TCE) treatment via cometabolism. An easy to maintain and operate laboratory scale packed‐bed biofilm reactor, featuring recycled gas and liquid effluent streams, was developed for continuous aerobic aqueous‐phase TCE biodegradation using Pseudomonas putida F1 as the biocatalyst and toluene as the primary substrate. The impacts of the influent toluene and TCE concentrations, flow rates, and recycle rates on TCE biodegradation were studied. Multivariate factors, including influent toluene and TCE concentrations, their interactions, and flow rates were further analyzed via a response surface model (RSM). Results showed that such bioreactor design eliminated oxygen limitation and removed or substantially reduced toluene competitive inhibition on TCE degradation. A TCE removal efficiency of >90% was achieved at an influent TCE concentration of ≤0.3 mg/L under an ˜8‐min mean hydraulic retention time. Toluene and TCE concentrations, in both linear and quadratic forms, were the most significant factors for TCE degradation, indicating both cell regenerating and inhibiting effects from toluene and a toxic effect from TCE. This RSM analysis has successfully identified an optimal influent toluene concentration path for each influent TCE concentration and provided insight for an extended TCE degradation at higher TCE concentrations.

An engineered toluene dioxygenase for a single step biocatalytical production of (-)-(1S,2R)-cis-1,2-dihydro-1,2-naphthalenediol

In modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.

Homogeneous catalytic hydrogenation of lipids in aqueous dispersions and bacterial cell membranes with an efficient water-soluble Pd(II)-sulfosalan catalyst, Na2[Pd(HSS)