Elucidation of the flavonoid catabolism pathway in Pseudomonas putida PML2 by comparative metabolic profiling.

Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3'-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.     

Phenylacetyl-Coenzyme A Is the True Inducer of the Phenylacetic Acid Catabolism Pathway in Pseudomonas putida U

Aerobic degradation of phenylacetic acid in Pseudomonas putida U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A [CoA] catabolon core). Induction of this route was analyzed by using different mutants specifically designed for this objective. Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

Drought-mitigating Pseudomonas putida Strain Modulates Polyamine Catabolism in Arabidopsis thaliana

Journal of Plant Growth Regulation - Plant growth-promoting rhizobacteria often play a significant role in alleviating drought stress in plants by colonizing the rhizosphere through myriad...     

Enzymes of the Tryptophan Synthetic Pathway in Pseudomonas putida

The first four enzymatic activities of the tryptophan synthetic pathway in Pseudomonas putida were found on separate molecules. Gel filtration and density gradient centrifugation experiments did not disclose any associations or aggregations among them. These findings contrast with the situation found in the enteric bacteria, where the first two activities are found in an aggregate and the third and fourth are catalyzed by a single enzyme. Tryptophan synthetase, the last enzyme of the pathway, consists of two dissociable components. The affinity of these components is less in P. putida than is the case in Escherichia coli.     

Catabolism of biphenyl by Pseudomonas putida BS 893 strain containing the biodegradation plasmid pBS241

The isolation and identification of biphenyl catabolism products in Pseudomonas putida BS 893 (pBS241) showed the presence of benzoic, m-hydroxybenzoic and cinnamic acids. The two latter compounds were not found in biphenyl degradation by other bacterial strains. P. putida BS 893 (pBS241) differed from other biphenyl-positive Pseudomonas strains in the enzyme activity. These differences may stem from peculiarities in the pathway of biphenyl catabolism controlled by plasmid pBS241.     

Regulation of the meta Cleavage Pathway for Benzoate Oxidation by Pseudomonas putida

Catechol or 2-hydroxymuconic semialdehyde cannot participate as functional inducers of the meta pathway for benzoate metabolism in Pseudomonas putida. Induction of the first two enzymes of the pathway must be mediated by benzoate, or its analogues, as primary substrate.     

Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-(13)C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter(-1). To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

The uptake of 2-ketogluconate by Pseudomonas putida

The uptake of 2-ketogluconate is inducible in Pseudomonas putida: 2-ketogluconate, glucose, gluconate, glycerol and glycerate were each good nutritional inducers of this ability. 2-Ketogluconate uptake obeyed saturation kinetics (apparent K _min 2-ketogluconate-grown cells was 0.4 mM). 2-Ketogluconate was transported against a concentration gradient, apparently in an unchanged state, and the process required metabolic energy, all of which indicate an active transport system.A number of independently isolated mutants with deranged activity of a common glucose-gluconate uptake system were found to be also defective in 2-ketogluconate transport. Strains unable to transport 2-ketogluconate which grew readily on glucose and gluconate were also isolated. These results suggest that 2-ketogluconate transport is governed by at least two genetic elements: one which is also required to take up glucose and gluconate and another which appears to be specific for 2-ketogluconate transport. Similarly glucose and gluconate transport appears to require at least one factor which is not necessary for 2-ketogluconate transport, as suggested by the lack of induction of the common glucose-gluconate uptake system by glycerol and glycerate, substrates which are good inducers of 2-ketogluconate uptake.Abbreviations CCCP carbonyl-cyanide-m-chlorophenyl-hydrazone - cpm radioactivity counts per minute - GGU glucose-gluconate uptake - PFU plaque forming units - U.V. ultraviolet Dedicated to Prof. Roger Y. Stainer on the occasion of his 60th birthday     

Catabolism of L-lysine by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PACI grows poorly on L-lysine as sole source of carbon but mutant derivatives which grow rapidly were readily isolated. Studies with one such mutant, P. aeruginosa PAC586, supported the existence of a route for L-lysine catabolism which differes from those reported previously in other species of Pseudomonas. The postulated route, the cadaverine or decarboxylase pathway, is initiated by the decarboxylation of L-lysine and involves the following steps: L-lysine leads to cadverine leads to I-piperideine leads 5-aminovalerate leads to glutarate semialdehyde leads glutarate. Evidence for this pathway is based on the characterization of the pathway reactions and the induction of the corresponding enzymes by growth on L-lysine. The first three enzymes were also induced by growth on cadaverine and to a lesser extent by 5-aminovalerate. No evidence was obtained for the presence of pathways involving L-lysine 2-monooxygenase or L-pipecolate dehydrogenase, but another potential route for L-lysine catabolism initiated by L-lysine 6-aminotransferase was detected. Studies with mutants unable to grow on L-lysine supported the existence of more than one catabolic pathway for L-lysine in this organism and indicated that all routes converge on a pathway for glutarate catabolism which generates acetyl-CoA. Pipecolate catabolism also appeared to converge on the glutarate pathway in P. AERUGINOSA. The results suggested that the growth rate of the parental strain is limited by the rate of transport and/or decarboxylation of L-lysine. The cadaverine pathway was present, but not so highly induced, in the parental strain P. aeruginosa PACI. Pseudomonas fluorescens contained enzymes of both the cadaverine (decarboxylase) and oxygenase pathways, strains of P. putida (biotypes A and B) contained enzymes of the oxygenase pathway but not the decarboxylase pathway and P. multivorans appeared deficient in both. All these species possessed L-lysine aminotransferase activity.     

Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl-PHA titre (g l⁻¹) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compounds and lignin.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Transport of succinate by Pseudomonas putida

Induced succinate uptake and transport (defined as transport of a compound followed by its metabolism and transport in the absence of subsequent metabolism) by Pseudomonas putida are active processes resulting in intracellular succinate concentrations 10-fold that of the initial extracellular concentration. Uptake was studied with the wild-type strain P. putida P2 and transport with a mutant deficient in succinate dehydrogenase activity. Addition of succinate, fumarate, or malate to the growth medium induces both processes above a basal level. Induction is dependent on protein synthesis and subject to catabolite repression. When extracts of induced and noninduced wild-type cells were assayed for succinate dehydrogenase, fumarase, and malate dehydrogenase only malate dehydrogenase increased in specific activity. Transport is inhibited by iodoacetamide, KCN, NaN₃, and 2,4-dinitrophenol and shows pH and temperature optima of 6.2 and 30 °C. Kinetic parameters are: basal uptake (cells grown on glutamate) K_m 11.6 μm, v 0.32 nmoles per min per mg dry cell mass; induced uptake (cells grown on succinate plus NH₄Cl) K_m 12.5 μm, v 5.78 nmoles per min per mg dry cell mass; induced transport (cells grown on nutrient broth plus succinate) K_m 10 μm, V 0.98 nmoles per min per mg dry cell mass. It was not possible to determine the kinetic parameters of basal transport. Malate and fumarate were the only compounds exhibiting competitive inhibition of uptake and transport suggesting common transport system for all three compounds. The K_i values for competitive inhibition and the K_m for succinate indicate the order of affinity for both uptake and transport are succinate > malate > fumarate. Data from kinetic parameters of uptake and transport and studies on succinate metabolism provide evidence consistent with concurrent increases in transport and metabolism to account for induced succinate uptake by P. putida.     

Degradation of Acetonitrile by Pseudomonas putida

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K_m of 40.61 mM. Mass balance studies with [¹⁴C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as ¹⁴CO₂ and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.