title: Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.
abstract: The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.
authors.affiliation: Department of Bioresource Science, Ibaraki University College of Agriculture, Ami, Ibaraki, 300-0393, Japan.
authors.forename: Takamasa
authors.initials: T
authors.lastname: Miura
authors.forename: Akito
authors.initials: A
authors.lastname: Nishizawa
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Nishizawa
authors.forename: Munehiko
authors.initials: M
authors.lastname: Asayama
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Shirai
mesh_terms: D001287:Attachment Sites, Microbiological; D002876:Chromosomes, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017906:Siphoviridae / Q000201:enzymology* / Q000235:genetics; D046939:Synechocystis / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics
chemical_list: D014764:Viral Proteins; D019426:Integrases