Constitutive Trichloroethylene Degradation Led by tac Promoter Chromosomally Integrated Upstream of Phenol Hydroxylase Genes of Ralstonia sp. KNl and ItsNucleotide Sequence Analysis
Nakamura. K Ishida. H Iizumi. T
Journal of bioscience and Bioengineering ; 2000 [Vol.89] Pages:47-54
Abstract
Ralstonia sp. KNl-1OA 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 IuxAB-encoding fragment was integrated downstream of phyE by homologous recombination in strain KNl-lOA, obtaining strain KNl-lOA-LX. In the same manner, the luxAB-encodIng fragment was integrated into the chromosome of the wild-type strain, KNl. The resultant strain KNl-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 KNl-lOA-LX was markedly slower than that by the phenol-induced KNl-LX. These results suggest that an important gene for TCE degradation was not transcribed by Ptac in KNl-lOA-LX. The nucleotide sequence analysis showed the existence of a small gene, phy2, upstream of phyA, and Ptac was found to be integrated into the middle of phyZ in KNl-lOA-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.
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