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Nitroaromatics Are Substrates for the TOL Plasmid Upper-Pathway Enzymes
A Delgado1, M G Wubbolts, M A Abril
1Departamento de Bioquímica Vegetal, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, Apto. 419, 18080 Granada, Spain, and Department of Biochemistry, University of Groningen, Groningen, The Netherlands.
Applied and Environmental Microbiology
|January 1, 1992
Summary
Engineered Pseudomonas putida and Escherichia coli can oxidize nitrotoluenes using toluene monoxygenase (TMO). This study details the accumulation of benzyl alcohols and benzaldehydes, and the substrate specificity of related enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbial Biotechnology
Background:
- The TOL plasmid encodes enzymes for toluene degradation.
- Toluene monoxygenase (TMO) initiates toluene oxidation.
- Genetic engineering can modify microbial metabolic pathways.
Purpose of the Study:
- To investigate the functional expression of xylMA genes encoding TMO in heterologous hosts.
- To characterize the oxidation of nitrotoluenes and the accumulation of products.
- To determine the substrate specificity of TOL-encoded dehydrogenases and the XylR regulator.
Main Methods:
- Cloning and expression of xylMA genes in a broad-host-range plasmid.
- Culturing Pseudomonas putida and Escherichia coli with nitrotoluene substrates.
- Quantification of accumulated benzyl alcohols and benzaldehydes using chromatography.
- Enzyme assays to determine substrate recognition by dehydrogenases and XylR.
Main Results:
- Expression of TMO enabled oxidation of toluene, m-nitrotoluene, and p-nitrotoluene in both hosts.
- Benzyl alcohols accumulated to approximately 80 muM, while benzaldehydes showed steady accumulation over 24 hours.
- TOL-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase accepted nitro-substituted compounds.
- The XylR regulator did not recognize nitro-substituted toluenes as effectors.
Conclusions:
- Heterologous expression of TMO facilitates nitrotoluene oxidation in P. putida and E. coli.
- The downstream enzymes of the TOL pathway exhibit broad substrate specificity for nitroaromatics.
- XylR's effector recognition is specific and does not extend to nitro-substituted toluenes, indicating pathway regulation differences.