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This paper reports the first method for the construction of unmarked gene deletion mutants in the genus Rhodococcus. Unmarked deletion of the kstD gene, encoding 3-ketosteroid Δ 1 -dehydrogenase (KSTD1) in Rhodococcus erythropolis SQ1, was achieved using the sacB counter-selection system. Conjugative mobilization of the mutagenic plasmid from Escherichia coli S17-1 to R. erythropolis strain...