110NISR RESEARCH GRANT
2003 Research Grant

Cell Surface Engineering in Aspergillus oryzae
Harushi NAKAJIMA
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo
Research aims

Results

Cell surface engineering is an innovative approach expressing heterologous proteins in the surface of microorganisms that are expected to offer functional protein-coatedcells as novel biocatalysts. In vivo display of GFP, glucoamylase, antigens, and metal-adsorbing peptides weresuccessfully achieved in bacteria and yeast by expression offusion protein of functional peptides and cell surface anchor. However, cell surface expression system has not yet
developed in filamentous fungi because of lack of anchors.
Our main purpose is search for useful cell surface anchor
protein in filamentous fungus A. oryzae, which has been
widely used for Japanese traditional fermentative food production.

b -N-acetylglucosaminidase has GlcNAc oligomer hydrolyzing activity and has shown to be involved in the formation of septa in filamentous fungi and germ tube inyeast. The nagA gene of A. oryzae encodes a protein of 600
amino acids with considerable homology to b -N-acetylglucosaminidases of various bacteria and fungi. We observed
at least a part of cell surface localization of NagA-eGFP fusion on the cell surface of A. oryzae hyphae in the solid culture (data not shown). However, the cell surface absorbingstability of the fusion protein was not high.
Hydrophobins have characteristic hydrophathy patterns,
and contain eight conserved Cys residues. Cell surface ofaerial hyphae, conidia, fruiting bodies and spores of filamentous fungi and mushrooms are covered with hydrophobin. Hydrophobins are secreted from fungus cells
and self-assembled at the cell surface to form a hydrophobic layer. Based on the genome database of A. oryzae, weisolated possible hydrophobin encoding genes (hypA–E).
The HypA from A. oryzae is highly homologous with theconidial hydrophobin RodA in A. nidulans (Fig. 1).
By western blot analysis, we found the solid-state culture
specific expression of hypA and hypB mRNA. The transcriptional regulation of hypA and hypB genes suggests theaerial hyphae and conidia specific localization of hy-MethodsWe isolated nagA and five hyp genes encoding b -Nacetylglucosaminidase and hydrophobins, respectively,
which are considered to be localized to the cell surface ofA. oryzae. The fusion genes of NagA-eGFP and HypAeGFP were constructed and introduced into A. oryzae. Expression and localization of the fusion proteins were investigated by Western blot analysis and fluorescent microscope.

Fig. 1.

Amino acid alignment of hydrophobins (HypA–E) from A. oryzae and A. nidulans RodA. Conserved Cys residues are underlined.

111>300 kDa

100 kDa

27 kDaFig. 2. HypA-eGFP is specifically localized at the surface of conidiophore and conidium of A. oryzae. Bar: 20 m m (left), 5 m m (right)

drophobins. To investigate the localization of HypA protein, we constructed a plasmid carrying hypA-egfp fusion
gene driven by the hypA promoter and introduced into A.
oryzae niaD300.
The fluorescence in the hyphae of the transformant washardly observed in submerged cultures. On the other hand,significant fluorescence was observed in the cell wall ofaerial structures particularly the conidia (Fig. 2). eGFP
(27 kDa) is relatively large tag for HypA (16 kDa). Westernblot analysis on the A. oryzae transformant cells revealed
three signals. The 27 kDa band is considered to be degraded

Fig. 3. HypA-eGFP is self-assembled on the surface of A. oryzae. Western blot analysis using anti-GFP antibody on the A. oryzae cells expressing
the hypA-egfp gene on the solid-state medium.

product and the 100 kDa band may be HypA-eGFP oligomer.
The most significant band with size more than 300 kDa wasassumed to be the self-assembled HypA-eGFP (Fig. 3). Hydrophobins in general are predominantly present as assemblages that are completely insoluble in hot SDS solutions.
Our observations indicated that eGFP-tagged HypA wassuccessfully expressed and self-assembled specifically at
the surface of aerial hyphae and conidia. We expect thatHypA will be useful cell surface anchor for exhibiting thefunctional peptides on the surface of filamentous fungus A.
oryzae.