Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia.

• First report of a temperature-sensitive replicon functional in Gordonia & Rhodococcus. • Amino acids for temperature-sensitive phenotype were predicted. • Molecular dynamics simulation was used to assess interaction and stability. • Mutant replicons were constructed by site-directed mutagenesis. • The temperature sensitive plasmid can be used in genome engineering applications. Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera. [ABSTRACT FROM AUTHOR]