Expression of Rhodococcus opacus alkB Genes in Anhydrous Organic Solvents(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)

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Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasolinecontaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM109 showed that they encode functional alkane hydroxylases with a substrate range of C_5-C_<16>. Promoters of the alkB1 and alkB2 genes, designated P_<alkB1> and P_<alkB2>, respectively, were examined for activity in anhydrous bis (2-ethylhexyl) phthalate (BEHP) containing C_5-C_<16> n-alkanes. Two recombinant plasmids, pP_<alkB1>EGFP and pP_<alkB2> EGFP, were constructed by inserting the egfp gene downstream of P_<alkB1> and P_<alkB2>, respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP_<alkB1> EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C_8 C_<16> n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P_<alkB1> activity in anhydrous BEHP was noticeably different from that in 0.85% NaCI. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.
社団法人日本生物工学会の論文
2008-08-25