Document Detail

Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene.
MedLine Citation:
PMID:  11165359     Owner:  NLM     Status:  MEDLINE    
In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.
M A Haro; V de Lorenzo
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of biotechnology     Volume:  85     ISSN:  0168-1656     ISO Abbreviation:  J. Biotechnol.     Publication Date:  2001 Feb 
Date Detail:
Created Date:  2001-02-22     Completed Date:  2001-05-17     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8411927     Medline TA:  J Biotechnol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  103-13     Citation Subset:  IM    
Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049, Madrid, Spain.
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MeSH Terms
Alcohol Oxidoreductases / genetics,  metabolism
Aldehyde Oxidoreductases / genetics,  metabolism
Biodegradation, Environmental
Citric Acid / metabolism
DNA Transposable Elements / genetics
Environmental Pollutants / metabolism*
Genes, Bacterial
Genetic Engineering
Oxygenases / genetics,  metabolism
Pseudomonas aeruginosa / genetics*,  metabolism*
Toluene / analogs & derivatives*,  metabolism*
Reg. No./Substance:
0/DNA Transposable Elements; 0/Environmental Pollutants; 108-88-3/Toluene; 77-92-9/Citric Acid; 95-49-8/2-chlorotoluene; EC 1.1.-/Alcohol Oxidoreductases; EC 1.1.1.-/benzyl alcohol dehydrogenase; EC 1.13.-/Oxygenases; EC dioxygenase; EC 1.2.-/Aldehyde Oxidoreductases; EC dehydrogenase (NAD+)

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