Identification of a methanol-inducible promoter from Rhodococcus erythropolis PR4 and its use as an expression vector

摘要

The genus Rhodococcus exhibits a broad range of catalytic activity and is tolerant to various kinds of organic solvents. This property makes rhodococci suitable for use as a whole-cell catalyst. Various tools for genetic engineering have been developed to use Rhodococcus erythropolis as a host for bioconversion. In this study, we investigated the protein expression responses of R. erythropotts strains and found that isocitrate lyase production in R. erythropolis PR4 (ICL_(Re)) was induced by methanol. By analyzing the regulation mechanisms of icl_(Re) expression, the ~200-bp upstream region from the first nucleotide of the translation initiation codon of icl_(Re) was shown to be sufficient for the methanol-inducible expression. Also, the ~100-bp upstream region exhibited strong constitutive promoter activity by an unknown mechanism(s). By investigating proteins that bound to the upstream region of icl_(Re) in vitro, a RamB homologue of R. erythropo/is PR4 (RamB_(Re)) was identified. Moreover, 2 putative RamB_(Re) binding sites were identified in the upstream region of icl_(Re) through pull-down assays. A ramB_(Re) knockout experiment suggested that RamB_(Re) negatively controlled the expression of icl_(Re) and that RamB_(Re) regulation was dependent on the availability of a carbon source. On the basis of these findings, we were able to create novel methanol-inducible and strong constitutive expression vectors.