New Tools for Stable Highly Efficient Gene Expression in Rhodococcus spp.

A new Escherichia coli–Rhodococcus bireplicon plasmid pRY3-Rho-MCS, which is low-copy in Rhodococcus and multicopy in E. coli and stably inherited in Rhodococcus, was constructed on the basis of the previously used plasmid pRY16. The new plasmid is capable of conjugative transfer from E. coli to R. rhodochrous and R. qingshengii. The resulting R. rhodochrous and R. qingshengii strains with the new plasmid stably overproduced the green fluorescent protein turboGFP under the control of the corynebacterial P_tuf promoter. The efficiency of the target gene expression in the engineered strains was evaluated by flow cytofluorimetry and the possibility of using this technology in the search for highly active promoters for target gene expression in Rhodococcus was demonstrated. The instability of inheritance of the original plasmid pRY16 in R. rhodochrous cells is potentially suitable for the development of CRISPR/Cas-based methods for genome editing of Rhodococcus spp. It is concluded that the obtained tools, the plasmid pRY3-Rho-MCS and the promoter P_tuf, have a significant potential for application in biotechnology, including the development of biocatalysts, as well as producers of bacterial proteins that are difficult to synthesize in E. coli and other model microorganisms.