|Budget Amount *help
¥7,100,000 (Direct Cost : ¥7,100,000)
Fiscal Year 2005 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 2004 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 2003 : ¥3,700,000 (Direct Cost : ¥3,700,000)
Genes encoding catechol 1,2-dioxygenase, cis, cis-muconate cycloisomerase, and muconolactone isomerase, which were constitutively synthesized and involved in the aniline metabolism of Rhodococcus sp. AN-22, were cloned. Primer extension analysis revealed a promoter region which transcribed these genes. Two genes, cspA and capB2, encoding cold shock proteins, were also cloned from this strain, and their promoter regions containing a cspB1 gene were identified.
The transformation method of Rhodococcus sp. AN-22 was improved, and the transformation efficiency increased 15-fold compared with the previous procedures. Lethal genes encoding a transcriptional factor GATA-1 (lethal peptide) or an inhibitor against DNA gyrase were introduced downstream of the cspB1 promoter region, and lethal cassettes activated by a cold condition were constructed. The cassettes were introduced into Rhodococcus sp. AN-22, and the growth of transformants carrying the cassettes were examined at 15℃. The parent stain Rhodococcus sp. AN-22 showed good growth at 15℃; on the other hand, the transformants carrying the cassettes couldn't be grown at 15℃. However, when plates where the transformants were inoculated and didn't show growth at 15℃ were incubated at 30℃, the colonies of transformants appeared on the plates. These results show that the stain AN-22 didn't die out by expression of the lethal genes under the control of the cspB1 promoter although the growth of the strain AN-22 was repressed by their translational products.
To construct a strong lethal cassette, cloning a gene encoding the restriction endonuclease SfiI was attempted. When cloned Sill genes by PCR were sequenced, all genes had mutation, which led translation of the enzyme SfiI to flame shift. These observations show that transformants carrying an intact SfiI gene died out by the expression of the cloned gene.