Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methylhydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P.putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P.putida NCIMB 9866 were also plasmid encoded. The nucleotide sequences of the two flavoprotein genes from P.putida NCIMB 9869 and P.putida NCIMB 9866 (pchF69A and pchF66, respectively) were the same except for 5 bases out of 1,584, and the translated amino acid sequences were identical. The nucleotide sequences of the genes for the cytochrome subunits of PCMH from the two bacteria (pchC69A and pchC66) varied by a single nucleotide in their 303-base sequences, and the translated amino acid sequences differed by a single residue at position 41 (Asp in PchC69A and Ala in PchC66). Both cytochromes had 21-residue signal sequences, as expected for periplasmic proteins, and these sequences were identical. On the other hand, no signal sequences were found for the flavoproteins.pchF69A and pchC69A were expressed, separately or together, in Escherichia coli JM109 and P.putida RA4007, with active PCMH produced in both bacteria. The E. coli-expressed flavocytochrome was purified. Our studies indicated that the E.coli-expressed subunits were identical to the subunits expressed in P.putida NCIMB 9869: molecular weights, isoelectric points, UV-visible spectra, and steady-state kinetic parameters were the same for the two sets of proteins. The subunits readily associated upon mixing two crude extracts of E.coli, one extract containing PchC69A and the other containing PchF69A. The courses of association of PchC69A and PchF69A were essentially identical for pure E. coli-expressed subunits and pure P. putida 9869-expressed subunits. E. coli-expressed PchC69A and PchF69A contained covalently bound heme and covalently bound flavin adenine dinucleotide, respectively, as the proteins expressed in nature.     

Gentisic acid and its 3- and 4-methyl-substituted homologues as intermediates in the bacterial degradation of m-cresol, 3,5-xylenol and 2,5-xylenol

1. Intact cells of a non-fluorescent Pseudomonas grown with m-cresol, 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol or 2,3,5-trimethylphenol rapidly oxidized all these phenols to completion. 3-Hydroxybenzoate and 2,5-dihydroxybenzoate (gentisate) were also readily oxidized. 2. 3-Hydroxybenzoic acid and 2,5-dihydroxybenzoic acid were isolated as products of m-cresol oxidation by cells inhibited by alphaalpha'-bipyridyl. Alkyl-substituted 3-hydroxybenzoic acids and alkyl-substituted gentisic acids were formed similarly from 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol and 2,3,5-trimethylphenol. 3. When supplemented with NADH, not NADPH, extracts of cells grown with 2,5-xylenol catalysed the oxidation of all five phenols and accumulated the corresponding gentisic acids in the presence of alphaalpha'-bipyridyl. 4. Cells of a fluorescent Pseudomonas grown with m-cresol oxidized m-cresol, 3,5-xylenol and 3-ethyl-5-methylphenol to completion and oxidized 2,5-xylenol and 2,3,5-trimethylphenol partially. The oxidation product of 2,5-xylenol was identified as 3-hydroxy-4-methylbenzoic acid. In the presence of alphaalpha'-bipyridyl, 3-hydroxy-5-methylbenzoic acid and 3-methylgentisic acid were formed from 3,5-xylenol.     

A Nad/nadh-dependent dehydrogenase from Pseudomonas putida acting on salbutamol

An arylalcohol dehydrogenase has been isolated from Pseudomonas putida NCIMB 9869 and was used for biosensing of salbutamol. The N-terminal hexadecapeptide sequence showed some homology to the form A of p-cresol methylhydroxylase and no homology to any other protein sequence of the Swiss Protein Sequence Data Bank. © Rapid Science Ltd. 1998     

γ-Irradiation of malic acid in aqueous solutions

The -irradiation of malic acid in aqueous solutions was studied under initially oxygenated and oxygen-free conditions in an attempt to determine the possible interconversion of malic acid into other carboxylic acids, specifically those associated with Krebs cycle. The effect of dose on product formation of the system was investigated. Gas-liquid chromatography combined with mass spectrometry was used as the principal means of identification of the non-volatile products. Thin layer chromotography and direct probe mass spectroscopy were also employed.The findings show that a variety of carboxylic acids are formed, with malonic and succinic acids in greatest abundance. These products have all been identified as being formed in the -irradiation of acetic acid, suggesting a common intermediary. Since these molecules fit into a metabolic cycle, it is strongly suggestive that prebiotic pathways provided the basis for biological systems.     

In situ proton NMR analysis of alpha-alkynoate biotransformations. From 'invisible' substrates to detectable metabolites.

Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible.     

P-cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896.

Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.     

Specific deletion of a large segment of pRA500: a 3,5-xylenol degradative plasmid

3,5-Xylenol degradative plasmid, pRA500 (approximately 500 kilobase pairs, kb) carried by Pseudomonas putida NCIB 9869, also encodes resistance to inorganic mercuric ions (Hg^r). Following growth of Ps. putida on benzoate, p -cresol, p -hydroxybenzoate or protocatechuate, the frequency of loss of the 3,5-xylenol phenotype was 75–90%. The deletion of a large segment of approximately 350 kb from pRA500, to give rise to archetypal plasmid designated pRA502, occurred in such 3,5-xylenol-negative derivatives. During the conjugational transfer of pRA500 following selection of transconjugants for Hg^ronly, frequency of loss of 3,5-xylenol phenotype was approximately 60%. A high number of these 3,5-xylenol-negative transconjugants carried pRA502. A site-specific deletion is suggested in the formation of archetypal plasmid pRA502 by growth of Ps. putida on the above compounds and during transfer of pRA500.     

Regulation of enzymes of the 3,5-xylenol-degradative pathway in Pseudomonas putida: evidence for a plasmid.

Constitutive synthesis of enzymes responsible for methyl group oxidation in 3,5-xylenol degradation and an associated p-cresol methylhydroxylase in Pseudomonas putida NCIB 9869 was shown by their retention at high specific activities in cells transferred from 3,5-xylenol medium to glutamate medium. The specific activities of other enzymes of the 3,5-xylenol pathway declined upon removal of aromatic substrate, consistent with their inducible control. Specific activities of the methyl-oxidizing enzymes showed an eventual decline concomitant with a decrease in the fraction of bacteria capable of growth with 3,5-xylenol; a simultaneous loss of the ability to grow with m-hydroxybenzoate was also observed. The property of 3,5-xylenol utilization could be transferred to another strain of P. putida. It is proposed that enzymes of the 3,5-xylenol pathway and those for conversion of p-cresol to p-hydroxybenzoate are plasmid encoded, that the early methyl-oxidizing enzymes are expressed constitutively, and that the later enzymes are inducible.     

Production of methanol from aromatic acids by Pseudomonas putida.

When grown at the expense of 3,4,5-trimethoxybenzoic acid, a strain of Pseudomonas putida oxidized this compound and also 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4-dihydroxy-5-methoxybenzoic (3-O-methylgallic) acids; but other hydroxy- or methoxy-benzoic acids were oxidized slowly or not at all. Radioactivity appeared exclusively in carbon dioxide when cells were incubated with [4-methoxyl-14C]trimethoxybenzoic acid, but was found mainly in methanol when[methoxyl-14C]3-O-methylgallic acid was metabolized. The identity of methanol was proved by analyzing the product from [methoxyl-13C]3-O-methylgallic acid by nuclear magnetic resonance spectroscopy and by isolating the labeled 3,5-dinitrobenzoic acid methyl ester, which was examined by mass spectrometry. These results, together with measurements of oxygen consumed in demethylations catalyzed by cell extracts, showed that two methoxyl groups of 3,4,5-trimethoxybenzoate and one of syringate were oxidized to give carbon dioxide and 3-O-methylgallate. This was then metabolized to pyruvate; the other product was presumed to be the 4-methyl ester of oxalacetic acid, for which cell extracts contained an inducible, specific esterase. P. putida did not metabolize the methanol released from this compound by hydrolysis. Support for the proposed reaction sequence was obtained by isolating mutants which, although able to convert 3,4,5-trimethoxybenzoic acid into 3-O-methylgallic acid, were unable to use either compound for growth.     

Production of Lactones and Peroxisomal Beta-Oxidation in Yeasts

Abstract

Among aroma compounds interesting for the food industry, lactones may be produced by biotechnological means using yeasts. These microorganisms are able to synthesize lactones de novo or by biotransformation of fatty acids with higher yields. Obtained lactone concentrations are compatible with industrial production, although detailed metabolic pathways have not been completely elucidated. The biotransformation of ricinoleic acid into gamma-decalactone is taken here as an example to better understand the uptake of hydroxy fatty acids by yeasts and the different pathways of fatty acid degradation. The localization of ricinoleic acid beta-oxidation in peroxisomes is demonstrated. Then the regulation of the biotransformation is described, particularly the induction of peroxisome proliferation and peroxisomal beta-oxidation and its regulation at the genome level. The nature of the biotransformation product is then discussed (4-hydroxydecanoic acid or gamma-decalactone), because the localization and the mechanisms of the lactonization are still not properly known. Lactone production may also be limited by the degradation of this aroma compound by the yeasts which produced it. Thus, different possible ways of modification and degradation of gamma-decalactone are described.     

In silico assessment of the metabolic capabilities of an engineered functional reversal of the β-oxidation cycle for the synthesis of longer-chain (C≥4) products

The modularity and versatility of an engineered functional reversal of the β-oxidation cycle make it a promising platform for the synthesis of longer-chain (C≥4) products. While the pathway has recently been exploited for the production of n-alcohols and carboxylic acids, fully capitalizing on its potential for the synthesis of a diverse set of product families requires a system-level assessment of its biosynthetic capabilities. To this end, we utilized a genome scale model of Escherichia coli, in combination with Flux Balance Analysis and Flux Variability Analysis, to determine the key characteristics and constraints of this pathway for the production of a variety of product families under fermentative conditions. This analysis revealed that the production of n-alcohols, alkanes, and fatty acids of lengths C3–C18 could be coupled to cell growth in a strain lacking native fermentative pathways, a characteristic enabling product synthesis at maximum rates, titers, and yields. While energetic and redox constraints limit the production of target compounds from alternative platforms such as the fatty acid biosynthesis and α-ketoacid pathways, the metabolic efficiency of a β-oxidation reversal allows the production of a wide range of products of varying length and functionality. The versatility of this platform was investigated through the simulation of various termination pathways for product synthesis along with the use of different priming molecules, demonstrating its potential for the efficient synthesis of a wide variety of functionalized compounds. Overall, specific metabolic manipulations suggested by this systems-level analysis include deletion of native fermentation pathways, the choice of priming molecules and specific routes for their synthesis, proper choice of termination enzymes, control of flux partitioning at the pyruvate node and the pentose phosphate pathway, and the use of an NADH-dependent trans-enoyl-CoA reductase instead of a ferredoxin-dependent enzyme.     

Molecular characterization of Pseudomonas aeruginosa 2NR degrading naphthalene

Three naphthalene-degrading strains were isolated from compost, characterized by morphological and physiological properties and differentiated by 16S rDNA RFLP. During growth on naphthalene, Pseudomonas aeruginosa 2NR produced ortho catechol pathway intermediates and gentisic acid. The ability to accumulate and degrade gentisic acid shows that Ps. aeruginosa 2NR has a different salicylate pathway to that of the intensely studied Ps. putida NCIB 9816. Molecular analysis showed the presence both of genes of the upper naphthalene pathway and genes of the ortho and meta catechol pathways. The insertion of nagH and nagG, coding for salicylate 5-hydroxylase in Pseudomonas sp. U2, was absent in Ps. aeruginosa 2NR, as in Ps. putida NCIMB 9816.     

Evaluation of various carbon substrates for the biosynthesis of polyhydroxyalkanoates bearing functional groups by Pseudomonas putida

The ability of Pseudomonas putida to synthesize polyhydroxyalkanoate (PHA) from 36 different carboxylic acids containing various functional groups was examined. This bacterium did not utilize short carboxylic acids (C(4)-C(6)) containing bromine, methoxy, ethoxy, cyclohexyl, phenoxy, and olefin groups as the sole carbon substrate. No polymer was isolated from the cells grown with carboxylic acids bearing hydroxyl, amino, para-methoxyphenoxy, and para-ethoxyphenoxy groups regardless of the carbon substrate chain lengths used even when they were cofed with nonanoic acid. Of all the carbon substrates evaluated, only 6-para-methylphenoxyhexanoic acid, 8-para-methylphenoxyoctanoic acid, 8-meta-methylphenoxyoctanoic acid, 10-undecenoic acid, and 10-undecynoic acid supported both growth and the production of PHA containing the corresponding functional groups by P. putida. The present results indicate that the carbon availability of P. putida for growth and PHA production is significantly different from that of P. oleovorans.     

Determination of isokinetic ratios necessary for equimolar incorporation of carboxylic acids in the solid-phase synthesis of mixture-based combinatorial libraries

The methods used to study the relative reaction rates of 45 different aliphatic and aromatic carboxylic acids when coupled to resin-bound amino acid amides is described. Competition experiments involving the coupling of incoming carboxylic acids to resin-bound amino acid amides were performed. The relative composition of each N-acylated amino acid amide in the resulting mixtures was compared to controls prepared by physically mixing equal aliquots of individual compounds in order to study the relative reaction rates of the incoming carboxylic acids. The ratios of the incoming carboxylic acids were then iteratively adjusted to yield as close to equimolar products as possible. As expected, the steric and electronic nature of the incoming carboxylic acids was found to influence their relative reaction rates. The steric hindrance of the resin-bound amino acid appears to have a proportional effect on the reaction rates of the incoming carboxylic acids. N-acylated amino acid amides in the final mixtures, prepared using the final isokinetic ratios, were found to be approximately equimolar.     

Biodegradation of cyanide compounds by a Pseudomonas species (S1).

A Pseudomonas sp. (S1), isolated from soil by an enrichment technique was tested for its potential to degrade different cyanide compounds. Further, biodegradation/biotransformation of binary mixtures of the cyanide compounds by the culture was also studied. The results indicated that the culture could grow on the following nitriles by using them as carbon and nitrogen sources: acetonitrile, butyronitrile, acrylonitrile, adiponitrile, benzonitrile, glutaronitrile, phenylacetonitrile, and succinonitrile. Studies on the biodegradation of these cyanide compounds in binary mixtures showed that the presence of acrylonitrile or KCN delayed the degradation of acetonitrile in a mixture, while none of the other cyanide compounds affected the degradation of one another. The transformation products of the nitriles were their corresponding acids, and similarly, KCN was also directly transformed to formic acid. Studies on the transformation of these cyanide compounds showed that the rate of transformation of nitriles to their corresponding carboxylic acids was acrylonitrile > acetonitrile > adiponitrile > benzonitrile > KCN. This culture has the unique characteristic of transforming representatives of saturated aliphatic, aliphatic olefinic, aromatic, and aralkyl nitriles, as well as alkali cyanide, to their corresponding carboxylic acids.     

The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal

The nitrilase of Rhodococcus rhodochrous performs a one-step biotransformation of nitriles to their corresponding carboxylic acids. Application of a direct electric current moves the charged carboxylic acid towards an anode, across an anion exchange membrane, into a separate compartment. Cells encapsulated within alginate beads (2.9 mm diameter) for protection against the current biotransformed benzonitrile to benzoic acid with a 26% reduction in the biotransformation rate, from 0.054 mmol/min/g dcw with free cells to 0.040 mmol/min/g dcw with immobilised cells. When the electric current was applied, the biotransformation rate increased to 0.047 mmol/min/g dcw and product recovery increased from 19% to 79%.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp. NCIMB12038

Rhodococcus sp. NCIMB112038 can utilize naphthalene as its sole carbon and energy source. The gene encoding cis-naphthalene dihydrodiol dehydrogenase (narB) of this strain has been cloned and sequenced. Expression of NCIMB12038 cis-naphthalene dihydrodiol dehydrogenase was demonstrated in Escherichia coli cells. narB encodes a putative protein of 271 amino acids and shares 39% amino acid identity with the cis-naphthalene dihydrodiol dehydrogenase from Pseudomonas putida G7. Comparison of NarB with some putative cis-dihydrodiol dehydrogenases from Rhodococcus species revealed significant differences between these proteins. NarB together with two other proteins forms a new group of cis-dihydrodiol dehydrogenases.     

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.     

Biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of Pseudomonas oleovorans producing the TOL plasmid-specified enzyme toluate-1,2-dioxygenase.

The conversion of substituted benzoates into 1,2-cis-dihydroxycyclohexa-3,5-diene carboxylic acids (cis-diols) was effected by using Escherichia coli and Pseudomonas recombinants carrying the xylXYZ genes originating from the Pseudomonas putida mt-2 TOL plasmid, thus producing toluate-1,2-dioxygenase. Pseudomonas oleovorans GPo12 recombinants readily produced meta- and para-substituted cis-diols, but were limited in their oxidation of ortho-substituted substrates.