Discovery of a bacterial gene cluster for catabolism of the plant hormone indole 3-acetic acid

The isolation and annotation of an 8994-bp DNA fragment from Pseudomonas putida 1290, which conferred upon P. putida KT2440 the ability to utilize the plant hormone indole 3-acetic acid (IAA) as a sole source of carbon and energy, is described. This iac locus (for indole 3-acetic acid catabolism) was identified through analysis of a plasposon mutant of P. putida 1290 that was no longer able to grow on IAA or indole 3-acetaldehyde and was unable to protect radish roots from stunting by exogenously added IAA. The iac locus consisted of 10 genes with coding similarity to enzymes acting on indole or amidated aromatics and to proteins with regulatory or unknown function. Highly similar iac gene clusters were identified in the genomes of 22 bacterial species. Five of these, i.e. P. putida GB-1, Marinomonas sp. MWYL1, Burkholderia sp. 383, Sphingomonas wittichii RW1 and Rhodococcus sp. RHA1, were tested to confirm that bacteria with IAA-degrading ability have representatives in the Alpha-, Beta- and Gammaproteobacteria and in the Actinobacteria. In P. putida 1290, cat and pca genes were found to be essential to IAA-degradation, suggesting that IAA is channeled via catechol into the beta-ketoadipate pathway. Also contributing to the IAA degrading phenotype were genes involved in tricarboxylate cycling, gluconeogenesis, and carbon/nitrogen sensing.     

Initial reactions in the mineralization of 2-sulfobenzoate by Pseudomonas sp. RW611

Abstract Pseudomonas sp. strain RW611 utilized the ammonium salt of 2-sulfobenzoate as sole source of carbon, sulfur, nitrogen, and energy. The xenobiotic sulfo substituent was dioxygenolytically eliminated as sulfite, which was then slowly oxidized to sulfate. 2,3-Dihydroxybenzoate, which resulted from desulfonation underwent meta -cleavage, mediated by 2,3-dihydroxybenzoate 3,4-dioxygenase activity. This enzyme was inhibited by 3-chlorocatechol and 2,3,4-trihydroxybenzoate.     

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.     

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100°C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10² cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples. Received 28 February 1997/ Accepted in revised form 23 November 1997     

Hyaluronan catabolism: a new metabolic pathway

A new pathway of intermediary metabolism is described involving the catabolism of hyaluronan. The cell surface hyaluronan receptor, CD44, two hyaluronidases, Hyal-1 and Hyal-2, and two lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase, are involved. This metabolic cascade begins in lipid raft invaginations at the cell membrane surface. Degradation of the high-molecular-weight extracellular hyaluronan occurs in a series of discreet steps generating hyaluronan chains of decreasing sizes. The biological functions of the oligomers at each quantum step differ widely, from the space-filling, hydrating, anti-angiogenic, immunosuppressive 10(4)-kDa extracellular polymer, to 20-kDa intermediate polymers that are highly angiogenic, immuno-stimulatory, and inflammatory. This is followed by degradation to small oligomers that can induce heat shock proteins and that are anti-apoptotic. The single sugar products, glucuronic acid and a glucosamine derivative are released from lysosomes to the cytoplasm, where they become available for other metabolic cycles. There are 15 g of hyaluronan in the 70-kg individual, of which 5 g are cycled daily through this pathway. Some of the steps in this catabolic cascade can be commandeered by cancer cells in the process of growth, invasion, and metastatic spread.     

A novel pathway for the biodegradation of 3-nitrotoluene in Pseudomonas putida

Abstract: 3-Nitrotoluene was degraded when incubated with the resting cells of Pseudomonas putida OU83. Most of the 3-nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3-aminotoluene (3-AT). A minor portion (30%) was degraded through a novel pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3-nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of a transient intermediary metabolite, hydroxynitroquinone, which was further degraded with the near stoichiometric release of nitrite into the medium. 3-Nitrotoluene-induced cells showed increased oxygen consumption with 3-nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as substrates in comparison to uninduced cells. Cell extracts prepared from strain OU83 contained benzylalcohol dehydrogenase and benzaldehyde dehydrogenase activities. The experimental evidence suggests a novel pathway for the degradation of 3-NT in which C-1 elimination is catalyzed by a cofactor-independent deformylase, rather than a decarboxylase or dioxygenase.     

Metabolism of 4-amino-3-hydroxybenzoic acid by Bordetella sp. strain 10d: A different modified meta-cleavage pathway for 2-aminophenols

Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2-oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloro-derivatives.     

A role for 3-hydroxy-3-methyl-glutaric acid in the biosynthesis of pseudomonic acid A

Abstract [3-¹⁴C]3-Hydroxy-3-methyl-glutaric acid (HMG) was incorporated (0.9%) into pseudomonic acid A when administered to a shaken flask fermentation of Pseudomonas fluorescens 4, 9 and 14 h after inoculation. [2-¹⁴C]-Mevalonic acid was not incorporated into pseudomonic acid. Experimental evidence supports the previous deduction that pseudomonic acid biosynthesis might directly involve HMG, an apparently unusual intermediary metabolite in prokaryotes.     

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO₂-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.     

A new abscisic acid catabolic pathway

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9'-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8'-carbon atom and at both the 8'- and 9'-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9'-methyl group, as well as at the 7'- and 8'-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9'-hydroxy ABA (9'-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9'-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.     

Production of L-2,3-butanediol by a new pathway constructed in Escherichia coli

AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most. SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future.     

Novel pathway for catabolism of the organic sulfur compound 3,3'-dithiodipropionic acid via 3-mercaptopropionic acid and 3-Sulfinopropionic acid to propionyl-coenzyme A by the aerobic bacterium Tetrathiobacter mimigardefordensis strain DPN7

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.     

Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.     

A novel pathway of glucose catabolism in Thiobacillus novellus

Phosphoketolase (E.C. 4.1.2.9) was found in crude extracts of Thiobacillus novellus (ATCC 8093) with a specific activity of 0.070 mol triose formed min^-1 (mg protein)^-1. The pH optimum, 6.0, temperature optimum, 43° C, and K _m , 4.27 mM, are in good agreement with values observed for phosphoketolase from other organisms.The level of phosphoketolase in lithotrophically grown T. novellus was observed to be much lower, 0.002 mol triose formed min^-1 (mg protein)^-1 than the level in heterotrophically grown cells. T. thioparus, a lithotroph, and T. intermedius, a mixotroph, were examined and found not to contain phosphoketolase. T. A2, a mixotroph reported to be very similar to T. novellus, was examined and found to have about 20% of the level of phosphoketolase as that seen in heterotrophically grown T. novellus.Fructose-6-phosphate was examined as a possible alternate substrate but was found not to be enzymatically cleaved in our system.It therefore appears that T. novellus utilizes a previously unknown combination of a partially complete hexose monophosphate pathway, the phosphoketolase reaction, and a partially complete Embden-Meyerhof-Parnas pathway, in conjunction with the Krebs Cycle, for glucose catabolism.     

A novel green enzymatic synthetic route of 2, 3-dihydroxybenzoic acid from glucose and CO2 fixation

The enzymatic carbon fixation is a promising approach to deal with greenhouse gas emission and is usually accompanied with energy consumption during the reduction of CO₂. As a very important route, the carboxylation can convert CO₂ to organic carbon without extra requirement of reduction power and is hoped as a greener solution, especially for some non-bulk chemicals, such as medical intermediates. Here, a concept-proof trail of green enzymatic process of conversing both of CO₂ and benzene to produce 2,3-dihydroxybenzoic acid (2,3-DHBA), which is the intermediate for fine chemicals, is introduced with O₂ from air and glucose. The results showed that the conversion catechol by 2, 3-dihydroxybenzoic acid decarboxylase (2,3-DHBD) alone was around 30 %, with an overall conversion from phenol of 2.4 %, which was limited by the in-situ production of catechol. This trail contributed a green enzymatic route for the production of 2,3-DHBA.     

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.