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

Abstract

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.

Modern Approaches to the Genome Editing of Antibiotic Biosynthetic Clusters in Actinomycetes

“…pSAM2 Site-Specific Recombination System В основе системы pSAM2 [78], как и в Cre-loxPподходе, лежит процесс сайт-специфической рекомбинации [79]. Но в данном случае специфичность обусловлена не активностью рекомбиназы, а определенными последовательностями в геномеattachment-сайтами: attP (в плазмиде pSAM2) и attB (в геномной ДНК бактерии) [80][81][82].…”

“…Такой подход применили для делеции биосинтетического кластера рифампицина, размером 90 т.п.н. в клетках A. mediterranei DSM 40773 [78]. К основным достоинствам данного метода относится то, что мутантные штаммы содержат дополнительные вставки в районе 30-40 п.н.…”

“…The pSAM2 system [ 78 ], like the Cre-loxP approach, is based on site-specific recombination [ 79 ]. But in this case, the specificity is associated not with recombinase activity, but with certain sequences in the genome— attachment sites attP (pSAM2 plasmid) and attB (genomic DNA of the bacterium) [ 80 , 81 , 82 ].…”

High-Yield Natural Vanillin Production by Amycolatopsis sp. after CRISPR-Cas12a-Mediated Gene Deletion

“…Some members of this genus are also well known for the production of the medically important antibiotics, rifamycin and vancomycin . Although genetic manipulation tools have been developed for Amycolatopsis spp., including a site-specific recombination system, shuttle vector, suicide vectors, and reporter genes, − compared with that of other actinobacterial genera such as Streptomyces , , efficient genome editing of Amycolatopsis spp. remains challenging.…”

“…Nevertheless, the entire process of two-step homologous recombination, including gene editing and plasmid curing, is still very time-consuming. Use of the pSAM2 site-specific recombination system to perform antibiotic resistance markerless gene deletion and large-fragment DNA knockout in Amycolatopsis spp . was successful, but a “scar”, a short (33 bp) section of residual DNA, remained on the genome, and this would impede the introduction of new genes into other targeted sites.…”