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Methods

Modified Cre-loxP Recombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue

Osamu Mizutani, Kazuo Masaki, Katsuya Gomi, Haruyuki Iefuji
Osamu Mizutani
aNational Research Institute of Brewing, Hiroshima, Japan
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Kazuo Masaki
aNational Research Institute of Brewing, Hiroshima, Japan
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Katsuya Gomi
bLaboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
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Haruyuki Iefuji
aNational Research Institute of Brewing, Hiroshima, Japan
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DOI: 10.1128/AEM.00080-12
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ABSTRACT

Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.

FOOTNOTES

    • Received 12 January 2012.
    • Accepted 30 March 2012.
    • Accepted manuscript posted online 13 April 2012.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00080-12.

  • Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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Modified Cre-loxP Recombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue
Osamu Mizutani, Kazuo Masaki, Katsuya Gomi, Haruyuki Iefuji
Applied and Environmental Microbiology May 2012, 78 (12) 4126-4133; DOI: 10.1128/AEM.00080-12

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Modified Cre-loxP Recombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue
Osamu Mizutani, Kazuo Masaki, Katsuya Gomi, Haruyuki Iefuji
Applied and Environmental Microbiology May 2012, 78 (12) 4126-4133; DOI: 10.1128/AEM.00080-12
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