Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System.

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces . We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis , expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR . We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis , for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis .