Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identifikacija i karakterizacija novih gena koji pripadaju putevima mikrobiološke razgradnje aromatičnih jedinjenja je od velikog značaja, jer su se pokazali kao izuzetno dobri biokatalizatori. U ovoj studiji, korišćenjem PCR metodologije, analizirano je prisustvo pet različitih gena iz biodegradativnog puta aromatičnih jedinjenja među 19 sredinskih izolata sa sposobnošću razgradnje širokog spektra aromatičnih jedinjenja. U slučaju 4-oksalokrotonat tautomeraze i toluen dioksigenaze, koji su detektovani kod većine sredinskih izolata, sekvence fragmenata su ukazivale na veoma ograničen diverzitet ova dva gena i visoku homologiju sa već poznatim sekvencama opisanim kod vrsta roda Pseudomonas. Korišćenjem degenerisanih prajmera konstruisanih na osnovu poznatih katehol-i naftalendioksigenaznih gena vrlo mali broj fragmenata je amplifikovan kod sredinskih izolata. Samo dve katehol 2,3-dioksigenaze iz dva izolata roda Bacillus su sekvenciranjem ukazale na različitost u odnosu na poznate sekvence, a pokazale međusobnu sličnost od 80-90%. Potencijalno tri nove katehol 1,2-dioksigenaze su identifikovane kod Bacillus sp. TN102, Gordonia sp. TN103 i Rhodococcus sp. TN112. Visok stepen homologije tautomeraza i toluen dioksigenaza među sredinskim izolatima izolovanim iz zagađene sredine ukazuje na horizontalni transfer gena, dok je ograničen uspeh u detektovanju preostala tri gena ukazao na potencijal da se među ovim izolatima mogu naći nove varijante gena iz puteva razgradnje aromatičnih jedinjenja.Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Limited Aromatic Pathway Genes Diversity Amongst Aromatic Compound Degrading Soil Bacterial Isolates

Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterised corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes

Synthesis of gamma-nitroaldehydes containing quaternary carbon in the alpha-position using a 4-oxalocrotonate tautomerase whole-cell biocatalyst

Synthesis of gamma-nitroaldehydes from branched chain aldehydes and a range of alpha,beta-unsaturated nitroalkenes was achieved by a whole-cell biocatalytic reaction using 4-oxalocrotonate tautomerase as catalyst. Under mild conditions, cyclic and acyclic branched aldehydes were converted into synthetically valuable quaternary carbon containing gamma-nitroaldehydes. The yields of the desired products were influenced by reaction condition parameters such as organic solvent, temperature and pH. The whole-cell biocatalytic approach to the generation of alpha,alpha-substituted gamma-nitroaldehydes was compared to the organocatalytic approach involving the lithium salt of phenylalanine as a catalyst. As the resulting gamma-nitroaldehydes exhibited moderate antifungal activity and mild in vitro cytotoxicity against human fibroblasts (0.2-0.4 mM) they could further be examined as potentially useful pharmaceutical synthons

Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes. [Projekat Ministarstva nauke Republike Srbije, br. 173048

Synthesis of gamma-nitroaldehydes containing quaternary carbon in the alpha-position using a 4-oxalocrotonate tautomerase whole-cell biocatalyst

Synthesis of gamma-nitroaldehydes from branched chain aldehydes and a range of alpha,beta-unsaturated nitroalkenes was achieved by a whole-cell biocatalytic reaction using 4-oxalocrotonate tautomerase as catalyst. Under mild conditions, cyclic and acyclic branched aldehydes were converted into synthetically valuable quaternary carbon containing gamma-nitroaldehydes. The yields of the desired products were influenced by reaction condition parameters such as organic solvent, temperature and pH. The whole-cell biocatalytic approach to the generation of alpha,alpha-substituted gamma-nitroaldehydes was compared to the organocatalytic approach involving the lithium salt of phenylalanine as a catalyst. As the resulting gamma-nitroaldehydes exhibited moderate antifungal activity and mild in vitro cytotoxicity against human fibroblasts (0.2-0.4 mM) they could further be examined as potentially useful pharmaceutical synthons