Degradation of 2,4,6-trichlorophenol by Phanerochaete chrysosporium: involvement of reductive dechlorination

Abstract

Under secondary metabolic conditions, the lignin-degrading basidiomycete Phanerochaete chrysosporium mineralizes 2,4, 6-trichlorophenol. The pathway for the degradation of 2,4, 6-trichlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway is initiated by a LiP- or MnP-catalyzed oxidative dechlorination reaction to produce 2,6-dichloro-1,4-benzoquinone. The quinone is reduced to 2,6-dichloro-1,4-dihydroxybenzene, which is reductively dechlorinated to yield 2-chloro-1,4-dihydroxybenzene. The latter is degraded further by one of two parallel pathways: it either undergoes further reductive dechlorination to yield 1, 4-hydroquinone, which is ortho-hydroxylated to produce 1,2, 4-trihydroxybenzene, or is hydroxylated to yield 5-chloro-1,2, 4-trihydroxybenzene, which is reductively dechlorinated to produce the common key metabolite 1,2,4-trihydroxybenzene. Presumably, the latter is ring cleaved with subsequent degradation to CO2. In this pathway, the chlorine at C-4 is oxidatively dechlorinated, whereas the other chlorines are removed by a reductive process in which chlorine is replaced by hydrogen. Apparently, all three chlorine atoms are removed prior to ring cleavage. To our knowledge, this is the first reported example of aromatic reductive dechlorination by a eukaryote.

FIG. 1

Effect of nitrogen concentration on the mineralization of ¹⁴C-labeled 2,4,6-TCP. Six-day-old stationary cultures of P. chrysosporium containing 1.2 (○) or 12 (•) mM ammonium tartrate were inoculated with radiolabeled substrate as described in the text. Flasks were purged with O₂, the evolved ¹⁴CO₂ was trapped, and the radioactivity was counted as described in the text.

FIG. 2

Metabolites identified from the P. chrysosporium degradation of 2,4,6-TCP and pathway intermediates. Cultures were incubated with either 2,4,6-TCP (I) or separately with the intermediate III, IV, VIII, or X and then extracted. Subsequently, products were analyzed as described in the text. HPLC was used to determine the yields of quinones. GC was used to determine the yields of other compounds. Percentages of substrates remaining or percent yields of products after incubation for 3 h (in brackets) or incubation for 6 h (in parentheses) are indicated. t, trace.

FIG. 3

Products identified from the oxidation of 2,4,6-TCP (I) and 2,6-dichloro-1,4-dihydroxybenzene (III) by purified MnP and LiP. Reaction conditions and identification of products were as described in the text. Percentages of substrate remaining and product yields (in parentheses) were determined by HPLC.

FIG. 4

Proposed pathway for the degradation of 2,4,6-TCP (I) by P. chrysosporium.