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Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803

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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.

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Acknowledgments

We thank T. Kondo (Nagoya University, Aichi, Japan) for providing us with the plasmid pOXL6803-2.

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Author notes

  1. Takamasa Miura

    Present address: Biological Resource Center, National Institute of Technology and Evaluation, Tokyo, Japan

  2. Akito Nishizawa

    Present address: Genaris, Inc., Yokohama, Kanagawa, Japan

Authors and Affiliations

  1. Department of Bioresource Science, Ibaraki University College of Agriculture, Ami, Ibaraki, 300-0393, Japan

    Takamasa Miura, Akito Nishizawa, Tomoyasu Nishizawa, Munehiko Asayama & Makoto Shirai

  2. Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, 113-8657, Japan

    Hideo Takahashi

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  1. Takamasa Miura
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  2. Akito Nishizawa
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  4. Munehiko Asayama
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  5. Hideo Takahashi
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Correspondence to Tomoyasu Nishizawa.

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Communicated by S. Hohmann.

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Miura, T., Nishizawa, A., Nishizawa, T. et al. Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803. Mol Genet Genomics 289, 615–623 (2014). https://doi.org/10.1007/s00438-014-0838-0

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  • DOI: https://doi.org/10.1007/s00438-014-0838-0

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