Hiroaki Ishida

Constitutive trichloroethylene degradation led by tac promoter chromosomally integrated upstream of phenol hydroxylase genes of Ralstonia sp. KN1 and its nucleotide sequence analysis

Ralstonia sp. KN1-10A is a strain capable of degrading trichloroethylene (TCE) constitutively due to the tac promoter (Ptac) integrated upstream of the phenol hydroxylase genes (phy) in its chromosome. The expression of Ptac was analyzed using luxAB of Vibrio harveyi as a reporter. After determining the nucleotide sequence of phyABCDE required for TCE degradation, a luxAB-encoding fragment was integrated downstream of phyE by homologous recombination in strain KN1-10A, obtaining strain KN1-10A-LX. In the same manner, the luxAB-encoding fragment was integrated into the chromosome of the wild-type strain, KN1. The resultant strain KN1-LX was used to analyze the gene expression caused by phenol induction. The expression induced by Ptac was compared to that by phenol induction. Although the level of luxAB expression led by Ptac was almost equal to that induced by phenol, the TCE degradation rate by the Ptac-carrying KN1-10A-LX was markedly slower than that by the phenol-induced KN1-LX. These results suggest that an important gene for TCE degradation was not transcribed by Ptac in KN1-10A-LX. The nucleotide sequence analysis showed the existence of a small gene, phyZ, upstream of phyA, and Ptac was found to be integrated into the middle of phyZ in KN1-10A-LX. The effect of phyZ on TCE degradation was examined by using recombinant strains expressing phyABCDE with or without phyZ in a plasmid. The coexistence of phyZ markedly accelerated TCE degradation. Through an exhaustive expression analysis, it was demonstrated that the chromosomal integration of Ptac was a very attractive method for high and stable production of phenol hydroxylase for TCE degradation.

Trichloroethylene degradation by Ralstonia sp. KN1-10A constitutively expressing phenol hydroxylase: Transformation products, NADH limitation, and product toxicity

Ralstonia sp. KN1-10A, which was constructed by inserting the tac promoter upstream of the phenol hydroxylase (PH) gene in the chromosomal DNA of the wild-type strain, Ralstonia sp. KN1, is a useful recombinant strain for eliminating trichloroethylene (TCE) from contaminated sites because it exhibits constitutive TCE oxidation activity. During TCE degradation by Ralstonia sp. KN1-10A, noxious chlorinated compounds, such as dichloroacetic acid, trichloroacetic acid, 2,2,2-trichloroethanol, and chloral, were not detected, and more than 95% of chlorine in TCE was released as chloride ions. Among the possible TCE transformation products, only carbon monoxide was detected, and its conversion percentage was 7 mol%. The addition of formate, which Ralstonia sp. KN1-10A could use as an exogenous electron donor, did not enhance the TCE degradation performance, suggesting that NADH depletion did not limit the degradation. The phenol degradation activity of Ralstonia sp. KN1-10A that previously degraded TCE was not markedly lower than that of cells not exposed to TCE, suggesting that Ralstonia sp. KN1-10A was not susceptible to product toxicity associated with TCE degradation. Furthermore, to clarify the mechanisms underlying TCE degradation by PH from Ralstonia sp. KN1, this enzyme was compared with another enzyme, a hybrid aromatic ring dioxygenase exhibiting a high TCE degradation activity in Escherichia coli and Pseudomonas sp. The initial TCE degradation rate of Ralstonia sp. KN1 (pKTP100), which produced PH, was 1 50 lower than that of Ralstonia sp. KN1 (pKTF200), which produced the hybrid aromatic ring dioxygenase. However, because of its lower product toxicity, the strain producing PH could degrade 2.3 times more TCE than that generated by the strain producing the hybrid aromatic ring dioxygenase.