Abstract
Previously we reported that double disruption of the proteinase genes (tppA and pepE) improved heterologous protein production by Aspergillus oryzae (Jin et al. Appl Microbiol Biotechnol 76:1059–1068, 2007). Since A. oryzae has 134 protease genes, the number of auxotrophy in a single host is limited for multiple disruptions of many protease genes. In order to rapidly perform multiple gene disruptions in A. oryzae, we generated the marker recycling system in highly efficient gene-targeting background. A. oryzae ligD gene homologous to Neurospora crassa mus-53 gene involved in nonhomologous chromosomal integration was disrupted, followed by disruption of the pyrG gene for uridine/uracil auxotroph. We further performed successive rounds of gene disruption (tppA and pepE) by the pyrG marker with high gene-targeting efficiency allowed by the ΔligD background. After each disruption process the pyrG marker was excised by the direct repeats consisting of ~300 bp upstream flanking region of the target gene, resulting in no residual ectopic/foreign DNA fragments in the genome. Consequently, we succeeded to breed the double proteinase gene disruptant (ΔtppA ΔpepE) applicable to further sequential gene disruptions in A. oryzae.
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Acknowledgements
We thank to Yukiko Oshima for experimental help. This study was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Supplementary Fig. 1
Southern analysis of the ligD disruptants. The genomic DNAs were digested with BamHI and SmaI. The boxed regions are the flanking regions used for disruption of the ligD gene. Four strains were analyzed, confirming that the strains (lanes 1 and 3 in the panels) exhibited the expected band pattern for disruption of the ligD gene. “P” represents the parental strain (NSAR1) (PDF 79 kb)
Supplementary Fig. 2
Southern analysis of the pyrG disruptant. The genomic DNAs were digested with PstI and XhoI. The flanking regions used for disruption of the pyrG gene are boxed. All 4 strains analyzed in the panel (lanes 1 to 4) exhibited the expected band pattern for disruption of the pyrG gene. “P” represents the parental strain (NSR-ΔlD2) (PDF 182 kb)
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Maruyama, JI., Kitamoto, K. Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (ΔligD) in Aspergillus oryzae . Biotechnol Lett 30, 1811–1817 (2008). https://doi.org/10.1007/s10529-008-9763-9
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DOI: https://doi.org/10.1007/s10529-008-9763-9