title: Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.
abstract: In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker.
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Silai
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Ban
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ebara
authors.affiliation: Applications Research Division, National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Mizutani
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shintani
authors.affiliation: Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Electronic address: katsuya.gomi.a6@tohoku.ac.jp.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Gomi
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics; D001236:Aspergillus oryzae / Q000235:genetics*; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D012641:Selection, Genetic
chemical_list: D005819:Genetic Markers; C045073:Cre recombinase; D019426:Integrases
keywords: Aspergillus oryzae; Cre/loxP; Kojic acid; Mutant lox; Pyrithiamine resistance; Selectable marker recycling; Transporter