Abstract
Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100.
Highlights
Because of widespread use of alkyl as well as halogen benzenes as reactants and solvents in chemical industry, they belong to the top ten of most frequently appearing contaminants in water and soil (UBA, 2013)
The major obstacle for degradation of toluene via ortho cleavage reaction is the fact that mainly 3-methylcatechol is formed as an intermediate of the oxidation of the ring. This intermediate is further transformed to 2-methyl-cis,cis-muconic acid followed by formation of 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-methylmuconolactone, 2-ML), which has frequently been described to be a dead-end metabolite
We previously described the strain Burkholderia fungorum FLU100 being able to mineralize all monohalogenated benzenes, benzene and toluene as pure substances by an ortho cleavage pathway (Strunk, 2000; 2007; Dobslaw, 2003; Strunk et al, 2006; Strunk and Engesser, 2013)
Summary
Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and monohalogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. 3methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100
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