Isolation of two plasmids, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400 and construction of a Rhodococcus–Escherichia coli shuttle vector

Abstract

With the aim of being able to co-express multiple genes, I searched for novel compatible plasmids and isolated two plasmid species, pRET1100 and pRET1200, from Rhodococcus erythropolis IAM1400. Sequencing analysis revealed that the pRET1100 plasmid is a double-stranded DNA molecule of 5444bp with two possible open reading frames (ORFs), repT and div, and three minor ORFs. The cryptic replication protein, RepT, is not highly homologous to those from other plasmids that have been reported. The Rhodococcus-Escherichia coli shuttle vector pRET1102 was transformed into R.erythropolis JCM2895 harboring the pRE2895 plasmid. The recombinant R.erythropolis JCM2895 harbored two plasmid species. These results suggest that plasmid derivatives of pRET1100 and pRE2895 are fully compatible in R.erythropolis. I determined the minimum region of pRET1100 required for autonomous replication in R.erythropolis and constructed a high-copy plasmid, pRET1129, in R.erythropolis.