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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 5, No. 2, 2002, pp. 162-172
Bioline Code: ej02027
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 5, No. 2, 2002, pp. 162-172

 en Engineering bacterial strains through the chromosomal insertion of the chlorocatechol catabolism tfdICDEF gene cluster, to improve degradation of typical bleached Kraft pulp mill effluent pollutants
Bobadilla, Roberto; Varela, Cristián; Céspedes, Ricardo & González, Bernardo

Abstract

Chloroaromatic pollutants from bleached Kraft pulp mill effluents (BKME) are difficult to degrade, because bacterial strains present in BKME aerobic treatments, only partially degrade these compounds, accumulating the corresponding chlorocatechol intermediates. To improve the catabolic performance of chlorocatechol-accumulating strains, we introduced, by chromosomal insertion, the tfdICDEF gene cluster from Ralstonia eutropha check for this species in other resources JMP134 (pJP4). This gene cluster allows dechlorination and channelling of chlorocatechols into the intermediate metabolism. Two bacterial strains, R. eutropha JMP222 and Pseudomonas putida check for this species in other resources KT2442, able to produce chlorocatechols from 3-chlorobenzoate (3-CB) were used. Acinetobacter lwoffii check for this species in other resources RB2 isolated from BKME by its ability to grow on guaiacol as sole carbon source and shown to be able to produce the corresponding chlorocatechols from the BKME pollutants 4-, and 5-chloroguaiacol, was also used. The tfdICDEF gene cluster was inserted in the chromosome of these strains using miniTn5-derived vectors that allow expression of the Tfd enzymes driven by the lacIq/Ptrc or tfdR/Ptfd-I regulatory systems, and therefore, responding to the inducers isopropyl-ß-D-thiogalactopyranoside (IPTG) or 3-CB, respectively. Crude extracts of cells from strains JMP222, KT2442 or RB2 engineered with the tfd genes, grown on benzoate and induced with IPTG or 3-CB showed Tfd specific activities of about 15% - 80% of that of the strain JMP134. Dechlorination rates for 3-CB or chloroguaiacols correlated with levels of Tfd enzymes. However, none of the strains containing the chromosomal copy of the tfdICDEF cluster grew on monochloroaromatics as sole carbon source. Experiments with BKME aerobic treatment microcosms showed that the catabolic performance of the engineered bacteria was also lower than the wild-type R. eutropha strain JMP134.

Keywords
chloroaromatics, chlorocatechols, metabolic complementation, Ralstonia eutropha check for this species in other resources , ortho ring cleavage, tfd check for this species in other resources genes

 
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