Characterization of a soil-derived bacterial consortium degrading 4-chloroaniline

A bacterial consortium comprising four different species was isolated from an Indonesian agricultural soil using a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources. The four species were identified as Chryseobacterium indologenes SB1, Comamonas testosteroni SB2, Pseudomonas corrugata SB4 and Stenotrophomonas maltophilia SB5. Growth studies on aniline and 4CA as single and mixed substrates demonstrated that the bacteria preferred to grow on and utilize aniline rather than 4CA, although both compounds were eventually depleted from the culture supernatant. However, despite 100 % disappearance of the parent substrates, the degradation of 4CA was always characterized by incomplete dechlorination and 4-chlorocatechol accumulation. This result suggests that further degradation of 4-chlorocatechol may be the rate-limiting step in the metabolism of 4CA by the bacterial consortium. HPLC-UV analysis showed that 4-chlorocatechol was further degraded via an ortho-cleavage pathway by the bacterial consortium. This hypothesis was supported by the results from enzyme assays of the crude cell extract of the consortium revealing catechol 1,2-dioxygenase activity which converted catechol and 4-chlorocatechol to cis,cis-muconic acid and 3-chloro-cis,cis-muconic acid respectively. However, the enzyme had a much higher conversion rate for catechol [156 U (g protein)−1] than for 4-chlorocatechol [17·2 U (g protein)−1], indicating preference for non-chlorinated substrates. Members of the bacterial consortium were also characterized individually. All isolates were able to assimilate aniline. P. corrugata SB4 was able to grow on 4CA solely, while S. maltophilia SB5 was able to grow on 4-chlorocatechol. These results suggest that the degradation of 4CA in the presence of aniline by the bacterial consortium was a result of interspecies interactions.

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Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas chlororaphis Strain UFB2

International Journal of Molecular Sciences ◽

10.3390/ijms20010061 ◽

2018 ◽

Vol 20 (1) ◽

pp. 61 ◽

Cited By ~ 3

Author(s):

Boitumelo Setlhare ◽

Ajit Kumar ◽

Mduduzi Mokoena ◽

Ademola Olaniran

Keyword(s):

Specific Activity ◽

Protein A ◽

Total Yield ◽

Gel Filtration ◽

Cell Extract ◽

Dimensional Structure ◽

Pseudomonas Chlororaphis ◽

Muconic Acid ◽

Crude Cell Extract ◽

Catechol 1,2 Dioxygenase

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver–Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.

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Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas Chlororaphis Strain UFB2

10.20944/preprints201810.0247.v1 ◽

2018 ◽

Author(s):

Boitumelo Setlhare ◽

Ajit Kumar ◽

Mduduzi P. Mokoena ◽

Ademola O. Olaniran

Keyword(s):

Specific Activity ◽

Protein A ◽

Total Yield ◽

Gel Filtration ◽

Cell Extract ◽

Dimensional Structure ◽

Pseudomonas Chlororaphis ◽

Muconic Acid ◽

Crude Cell Extract ◽

Catechol 1,2 Dioxygenase

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathway, respectively. The aim of the study was to purify, characterize and predict template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02 % by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and temperature 30 °C. The Lineweaver-Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O share 81% homology to homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. Characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.

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A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Applied and Environmental Microbiology ◽

10.1128/aem.00859-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4024-4030 ◽

Cited By ~ 63

Author(s):

Xinxiao Sun ◽

Yuheng Lin ◽

Qin Huang ◽

Qipeng Yuan ◽

Yajun Yan

Keyword(s):

Adipic Acid ◽

Scale Up ◽

Carbon Sources ◽

Shikimate Pathway ◽

Tryptophan Biosynthesis ◽

Regeneration System ◽

Muconic Acid ◽

Promising Alternative ◽

Content Type ◽

Catechol 1,2 Dioxygenase

ABSTRACTMuconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway inEscherichia colifor the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineeredE. colistrain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.

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Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01386-15 ◽

2015 ◽

Vol 81 (23) ◽

pp. 8037-8043 ◽

Cited By ~ 37

Author(s):

Sudeshna Sengupta ◽

Sudhakar Jonnalagadda ◽

Lakshani Goonewardena ◽

Veeresh Juturu

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Carbon Sources ◽

Raw Material ◽

Hydroxybenzoic Acid ◽

Muconic Acid ◽

Content Type ◽

E Coli ◽

Catechol 1,2 Dioxygenase ◽

K 12

ABSTRACTcis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain ofEscherichia coliK-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes,pobA,aroY, andcatA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed inE. colito establish the MA biosynthetic pathway.E. colinative genesubiC,aroFFBR,aroE, andaroLwere overexpressed and the genesptsH,ptsI,crr, andpykFwere deleted from theE. coligenome in order to increase the precursors of the proposed MA pathway. The final engineeredE. colistrain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.

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Faculty Opinions recommendation of GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1093774.548696 ◽

2007 ◽

Author(s):

Sjef Smeekens

Keyword(s):

G Protein ◽

Sugar Signaling ◽

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting ◽

G Protein Coupled

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Modelization of Nutrient Removal Processes at a Large WWTP Using a Modified ASM2d Model

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph15122817 ◽

2018 ◽

Vol 15 (12) ◽

pp. 2817 ◽

Cited By ~ 4

Author(s):

Jakub Drewnowski ◽

Jacek Makinia ◽

Lukasz Kopec ◽

Francisco-Jesus Fernandez-Morales

Keyword(s):

Nutrient Removal ◽

Carbon Sources ◽

Oxygen Demand ◽

Full Scale ◽

Biological Nutrient Removal ◽

Organic Substrates ◽

Modified Model ◽

Rate Limiting Step ◽

Uptake Rates ◽

Rate Limiting

The biodegradation of particulate substrates starts by a hydrolytic stage. Hydrolysis is a slow reaction and usually becomes the rate limiting step of the organic substrates biodegradation. The objective of this work was to evaluate a novel hydrolysis concept based on a modification of the activated sludge model (ASM2d) and to compare it with the original ASM2d model. The hydrolysis concept was developed in order to accurately predict the use of internal carbon sources in enhanced biological nutrient removal (BNR) processes at a full scale facility located in northern Poland. Both hydrolysis concepts were compared based on the accuracy of their predictions for the main processes taking place at a full-scale facility. From the comparison, it was observed that the modified ASM2d model presented similar predictions to those of the original ASM2d model on the behavior of chemical oxygen demand (COD), NH4-N, NO3-N, and PO4-P. However, the modified model proposed in this work yield better predictions of the oxygen uptake rate (OUR) (up to 5.6 and 5.7%) as well as in the phosphate release and uptake rates.

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Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12

Applied and Environmental Microbiology ◽

10.1128/aem.00685-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6606-6613 ◽

Cited By ~ 11

Author(s):

Dhan Prakash ◽

Ravi Kumar ◽

R. K. Jain ◽

B. N. Tiwary

Keyword(s):

Salicylic Acid ◽

Tricarboxylic Acid Cycle ◽

Sole Source ◽

Degradation Pathway ◽

Ring Cleavage ◽

Muconic Acid ◽

Content Type ◽

Nitrobenzoic Acid ◽

Catabolic Genes ◽

Catechol 1,2 Dioxygenase

ABSTRACTThe organismAcinetobactersp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidativeorthodehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and18O2indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by theorthoring cleavage catechol-1,2-dioxygenase tocis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA−derivative and a 2C4NBA+transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ∼55-kb transmissible plasmid present in RKJ12.

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