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. erythropolis strains and found that isocitrate lyase production in R. erythropolis PR4 (ICLRe) was induced by methanol. By analyzing the regulation mechanisms of icl(Re) expression, the similar to 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 similar to 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. erythropolis 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. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.