1784 Furthermore, recombinant A. oryzae strains have been developed for the production of heterogeneous and endogenous proteins, because this microorganism secretes large amounts of protein (Christensen et al. 1988; Iwashita 2002; Kitamoto 2002). However, to our knowledge, cell surface display systems in A. oryzae have only been developed using a glycosylphosphatidylinositol anchor protein (Adachi et al. 2008). Therefore, to expand the A. oryzae cell surface display system, the development of other anchor proteins for display on A. oryzae is needed. The site of the displayed protein and anchor protein fusion (i.e., N or C terminus) is one of the important factors in determining whether the target proteins are displayed without loss of function. In yeast and lactic acid bacteria cell surface display systems, it has been demonstrated that the activity of displayed proteins depends on the fusion site (N or C terminus) of the anchor proteins (Shigechi et al. 2004; Okano et al. 2008). α-Amylase from Streptococcus bovis 148 (Satoh et al. 1993) and lipase from Rhizopus oryzae show high activity when fused to the C terminus of the anchor protein, but poor activity when fused to the N terminus (Shigechi et al. 2004; Washida et al. 2001). For A. oryzae, there is currently no anchor protein that allows fusion of a target protein to either the N or C terminus. In this study, to expand the utility of A. oryzae, we established a novel cell surface display system that enables the target protein to fuse to both N and C termini of the anchor protein. We focused on the chitin-binding module (CBM) from Saccharomyces cerevisiae, which has high affinity to chitin (Kuranda and Robbins 1991). A. oryzae has a large amount of chitin on its cell surface (Seidl 2008; Higuchi et al. 2009), and therefore CBM should be suitable as an anchor protein which tightly binds to the A. oryzae cell wall. Using CBM as an anchor protein, we tested the displaying possibilities of green fluorescent protein (GFP) and triacylglycerol lipase from A. oryzae (tglA). Materials and methods Strains and media Escherichia coli NovaBlue (Novagen, Inc., Madison, WI) was used as the cloning host for recombinant DNA manipulations. The bacterium was grown in Luria–Bertani medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) containing 0.1 mg/ml of ampicillin. The A. oryzae niaD mutant (strain IF4), derived from wild-type A. oryzae OSI1031, was used as the expression host for the novel cell surface display system. Czapek–Dox (CD) medium plates (2% glucose, 0.3% NaNO3 (CD-NO3), 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, and 0.8 M NaCl, pH 6.0) containing 1.5% agar were used as the minimal Appl Microbiol Biotechnol (2010) 87:1783–1789 medium. The plate was used to select the fungal trans- formants. GPY medium (3% glucose, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 1% peptone, and 0.5% yeast extract, pH 6.0) was used for growing the A. oryzae IF4 and transformants. All transformants and wild-type A. oryzae used for all analyses in this study were cultivated in Sakaguchi flasks (500 ml) containing 100 ml of GPY medium. Construction of expression vectors and A. oryzae transformation A. oryzae transformation vectors were constructed using the pISI vector (Research Institute, Gekkeikan Sake Co., Kyoto, Japan), which contains the sodM promoter and glaB terminator from A. oryzae (Ishida et al. 2004). Polymerase chain reaction (PCR) amplification of DNA fragments was performed using KOD plus DNA polymerase (Toyobo, Osaka, Japan) according to the manufacturer's protocol. The pISI-GFP vector (Adachi et al. 2008) containing the tglA signal sequence from A. oryzae (Toida et al. 2000), the N28 sequence from R. oryzae (Hama et al. 2006; Hama et al. 2008), and the EGFP gene PCR amplified from pEGFP (Clontech Laboratory, Mountain View, CA) was used as the GFP secreting vector. The GFP anchoring expression vectors were constructed as follows. The DNA fragment encoding CBM was amplified by PCR using the genome from the S. cerevisiae W303-IB strain using the following primers, 5′-GCTAATGGAGCGGCCGCATCAGACAGT ACAGCTCGTACATTGGCTAAAGA-3′ and 5′-CCATA GGATATTTAAATCTAAAAGTAATTGCTTTCCAA ATAAGAGAAATT-3′ (underlined sequences indicate re- striction enzyme sites). The amplified fragment was digested with NotI and SwaI, and inserted into pISI-GF P-MP1 (Adachi et al. 2008). The resultant plasmid was designated as pISI-GFP-CBM. The DNA fragment encoding the last 15 bases of the N28 sequence, CBM, and the last 15 bases of GFP was amplified from the genome of the S. cerevisiae W303-IB strain with the following primers, 5′- A A C A G C G C C A A G C G T T G G C C AT C A G A C A G TA CAGCTCGTAC-3′ and 5′-GCCCTTGCTCACCATCATA TGAAAGTAATTGCTTTCCAAAT-3′. The amplified fragment was inserted into SalI-digested linear pISI-GFP using the In-Fusion enzyme (Takara Bio, Otsu, Japan) according to the manufacturer’s procedure. The resultant plasmid was designated as pISI-CBM-GFP. The tglA secreting and anchoring vectors were constructed as follows. The DNA fragment encoding the mature region of tglA from A. oryzae that was fused with the gene encoding the FLAG peptide tag at the C terminus was amplified from cDNA prepared from A. oryzae with the following primers, 5′-ACGCGTCG