Transformation of Chlorinated Benzenes and Toluenes by Ralstonia sp. Strain PS12 tecA (Tetrachlorobenzene Dioxygenase) and tecB (Chlorobenzene Dihydrodiol Dehydrogenase) Gene Products

Abstract

The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5␣ together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3-and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di-and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloroand 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.

Rational engineering of the regioselectivity of TecA tetrachlorobenzene dioxygenase for the transformation of chlorinated toluenes

“…Bacterial strains and plasmids used were Escherichia coli DH5a (Clonetech) as host for cloning; pBluescript II KS(+) (Stratagene); pSTE44, carrying the genes tecAB encoding tetrachlorobenzene dioxygenase (TecA) and dihydrodiol dehydrogenase (TecB) (Pollmann et al, 2001); pKPM1 was obtained by cloning a 3976 bp EcoRI fragment comprising the complete tecA1A2A3 genes from a digest of pSTE44 into the EcoRI site of pBluescript II KS(+); pKPM2, pKPM3, pKPM4, pKPM5 and pKPM6 were obtained by cloning the 1164 bp RsrII-and MluI-digested 1240 bp PCR products obtained from splicing by overlap extension (SOE)-PCR carrying mutations in F366W (pKPM2), F366Y (pKPM3), L272W (pKPM4), L272F (pKPM5) and F366L (pKPM6), respectively, into RsrII-and MluI-digested pKPM1; pKPM7, pKPM9, pKPM10, pKPM11 and pKPM12 were obtained by cloning a 1922 bp Eco47III fragment comprising the complete mutated tecA1 gene from pKPM2, pKPM3, pKPM4, pKPM5 and pKPM6 into the Eco47III site of pSTE44. Thus, pKPM7, pKPM9, pKPM10, pKPM11 and pKPM12 differ from pSTE44 only by the respective mutation.…”

“…The alcohol products were identified and quantified by comparison with authentic standards. Concentrations of dichloromethylcatechols and 2,4,5-trichlorobenzylalcohol were determined by HPLC analyses as described previously (Pollmann et al, 2001). Substrate transformation rates were expressed as the amount of product formed per time as determined by HPLC.…”

“…Similarly, the a-subunits have been reported to be dominantly responsible for the substrate specificities of biphenyl, naphthalene and nitrotoluene dioxygenases (Kauppi et al, 1998;Parales et al, 1998;Tan & Cheong, 1994). Besides dioxygenation of the aromatic nucleus, TecA catalyses mono-oxygenation of the methyl group of various chlorosubstituted toluenes, with the ratio of di-versus mono-oxygenation dependent on the substitution pattern (Lehning et al, 1997;Pollmann et al, 2001).…”

“…PS12 (Beil et al, 1997) is a broad specificity enzyme which catalyses the first step in the degradation of a wide variety of chlorinated benzenes and toluenes (Beil et al, 1997;Lehning et al, 1997;Pollmann et al, 2001;Sander et al, 1991). Like other dioxygenases acting on hydrophobic substrates, the enzyme is composed of an electron transport chain involving a reductase and a ferredoxin and the terminal dioxygenase consisting of aand b-subunits.…”

Biodegradation of 2,4-dichlorophenol using Mycoplana dimorpha extracts and evaluation of kinetic parameters

“…J. Biotechnol. indicates that immobilized crude extract degradation is much faster than microbial degradation (Axcell and Geary 1973;Grund et al, 1975;Hartmann et al, 1979;Chang et al, 1992;Spiess and Gorisch 1996;Beil et al, 1997;Mars et al, 1997;Beil et al, 1998;Pollmann et al, 2001;Alfreider et al, 2003;Rapp and Gabriel-Jurgens 2003;Manikandan et al, 2007a;Manikandan et al, 2007b). The similar trend of decomposition was observed with the initial concentrations of 60 and 80 ppm with the percentage degradation at the end of 30 min is 72% for 60 ppm and 75% for 80 ppm (Manikandan et al, 2007d).…”