Genomic deletions in Rhodococcus based on transformation of linear heterologous DNA

Abstract

Several genome engineering methods have been developed for Rhodococcus . However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

Microbial Musings – March 2021

“…While there are a variety of multistep methods for genetic manipulation that have been used across different species of this genus, here the authors find that a simple homologous recombination method like that developed by Datsenko and Wanner [22] for E. coli will work in Rhodococcus erythropolis [23]. The authors from the group of Preeti Srivastava at the Indian Institute of Technology, New Delhi, India, use this simple method, where designed linear DNA is electroporated into cells to select for mutants with kanamycin resistant replacements of certain genes they were interested in.…”