Degradation of toluene by ortho cleavage enzymes in B urkholderia fungorum FLU 100

Summary B urkholderia fungorum   FLU 100 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 FLU 100 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   FLU 100.