It is, however, more difficult to mutate multiple genes this way [10, 11].A BLAST search using the pyrG sequence Axitinib buy of A. oryzae S1 against the A. oryzae RIB40 genome database at the National Institute of Technology and Evaluation (NITE; http://www.bio.nite.go.jp/dogan) showed that pyrG is present as a single copy on chromosome 7 (contig SC011, location 2219667��2220565).In this study, a gene replacement cassette was constructed consisting of the native pyrG locus extending 1.5 to 2.0kb beyond the upstream and downstream termini of the pyrG coding region but lacking the open reading frame of the gene. Transformation of A. oryzae strain S1 with the gene replacement cassette generated 5-FOA-resistant uracil and uridine auxotrophs.
Putative pyrG��0 mutants were verified by comparing their growth on selective and nonselective media, PCR analyses of the pyrG region in the putative mutants and complementation with plasmids containing wild-type homologous and heterologous pyrG genes. Suitability of the pyrG��0 mutant as a host for heterologous protein production was assessed using strain GR6 harbouring a plasmid in which the pyrG and heterologous ��-galactosidase genes were integrated.2. Materials and Methods2.1. Fungus, Cultivation, and PlasmidsA. oryzae strain S1was obtained from the Fungal Stock Culture Collection of the Molecular Mycology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia. The fungus was maintained on a Potato Dextrose Agar (PDA; Difco, France) at 30��C and subcultured monthly.
Fungal cultivation was carried out by inoculating onto a fresh PDA plate. Cultures were incubated for 7 days at 30��C. Plasmid ANEp2 (Figure 1(a)) is a shuttle vector capable of autonomous replication in Aspergilla [12]. In addition to the pUC18 backbone ANEp2 carries the A. nidulans pyrG gene as a selectable marker, an expression cassette (GlaPr-lacA-GlaTt) that expresses an A. niger secreted ��-galactosidase, and the AMA1 sequence that supports autonomous replication in Aspergilli. ANEp2 was used to functionally test for complementation of uridine auxotrophy and reporter gene expression in an A. oryzae pyrG mutant. Plasmid pUC_pyrGAo containing the pyrG of the A. oryzae strain S1 was also used for complementation of uridine auxotrophy (Figure 1(b)) [13]. Escherichia coli strain DH5�� was used for DNA manipulation.
Figure 1Plasmids used for transformation. (a) Plasmid ANEp2 containing the pyrG gene of A. nidulans and a GlaPr-lacA-GlaTt chimera (b) plasmid pUC_pyrGAo containing pyrG (1,839bp) of A. oryzae strain S1.2.2. MediaPolypeptone dextrin (PD) medium containing 1.0% (w/v) polypeptone, 2.0% (w/v) glucose, 0.5% (w/v) KH2PO4, Brefeldin_A 0.1% (w/v) NaNO3, and 0.05% (w/v) MgSO4?7H2O was used for liquid cultivation of A. oryzae [14]. Czapek-Dox (CD) medium composed of 0.2% (w/v) NaNO3, 0.1% (w/v) K2HPO4, 0.05% (w/v) MgSO4?7H2O, 0.