51NISR Research GRANT
2009 Young Investigator Research Grant

Identification and Application of New Genes Involved in Glycosylation in Filamentous Fungi
Takuji OKA
Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University

Research aims

DNA fragments were combined as a template and the genereplacement cassette was amplified using primers F1 andR3 using Phusion DNA polymerase (Finnzymes). The amplified DNA fragments were then transformed into strain
AKU89 using the protoplast-PEG method (Fig. 1). Transformants were analyzed for correct gene replacement byPCR and Southern blot analysis.

Most of the enzymes from Aspergillus, including kojimold, are glycosylated, which improves both the functioning and efficacy of these enzymes. Although galactofuranose and glucose residues have been observed to be
attached to the O-glycans and N-glycans of proteins inAspergillus, the glycosyltransferase genes involved in thebiosynthesis of these sugar residues have not yet been identified and the function of these sugar moieties also is not yet
known1).
The ability of filamentous fungi to secrete large quantities of proteins means that they are well suited for use as
expression hosts for extragenous genes in industrial applications. However, since the sugar moieties of glycoproteinsfrom filamentous fungi are antigenic in humans, they cannot be used for medicinal applications, such as antibody
preparation. Consequently, a method for altering the structure of these sugar moieties needs to be developed so that aprotein expression system using filamentous fungi can bedeveloped.
We therefore sought to identify the genes involved in thebiosynthesis of these sugar moieties in filamentous fungi,and to elucidate the function of these sugar moieties in fungal growth and protein secretion.

To analyze the function of the putative glycosyltransferase genes, we disrupted each of the putative genes in A.
nidulans AKU89 by gene replacement with argB.
Strain DAN8677 formed a fragile colony after incubation
on minimal medium (MM medium) at 30°C for 3 days.
Growth defects was not almost recovered on MM medium
in the presence of 0.6 M KCl. The growth rate of strain
DAN8677 (0.24 mm/hr) was 66% of that for the wild-typestrain (0.35 mm/hr). The conidiation efficiency of strain
DAN8677 (6.6107/mm2) was reduced to approximately
10% of that for the wild-type strain (6.0108/mm2). Similarly, monosaccharide analysis of the cell wall revealed thatthe galactose residues in the DAN8677 stain were reduced
to 63.7% compared to the wild-type strain. It therefore
seems likely that the AN8677 gene is involved in theMethods

Table 1. List of putative glycosyltransferase genes

Using the Carbohydrate Active Enzymes (CAZy) database, approximately 90 putative glycosyltransferase genes
were identified in the Aspergillus nidulans genome2,3). Afterexcluding those genes having high sequence homology
with genes of known function in other organisms, 19 putative candidate glycosyltransferase genes remained (Table1). Each of the 19 glycosyltransferase genes were then disrupted and the resulting phenotypes were examined.
The DnkuB strain of A. nidulans (AKU89), which has ahigh gene-targeting frequency, was used as the host4). For
each of the 19 target genes, a gene replacement cassette, inwhich a selection marker gene (argB) was flanked by the5- and 3-flanking sequences of the target gene, was constructed by recombinant PCR. First, the 5- and 3- flanking
regions of the A. nidulans genomic DNA were amplified
using primer pairs FC/R1 and F3/RC, respectively, andargB of pDC1 (Fungal Genetics Stock Center) was amplified using primers F2 and R2. Then, after the three purified

Results

Systematic Name

GT family No.

AN2923
AN5058AN6460AN10604
AN2015AN5663AN8677
AN4824AN7535
AN8627
AN11144
AN11697
AN4092AN4738AN7483AN6571
AN6857AN0246
AN4084

GT25GT25GT25GT25GT31GT31GT31GT31GT31GT31GT31GT31GT69GT69GT69GT71GT71
GT90GT9052Fig. 1.

Strategy of the gene disruption

Conclusion
Of the 19 disruptants, a reduction in the growth rate andconidia formation was observed in strain DAN8677. In addition, no b 1,5-galactofuranose residues were observed in
the cell wall proteins of the mutant. These results demonstrated that the AN8677 gene is involved in the biosynthesis of b 1,5-galactofuranose residues.
References1)
2)

Fig. 2.

Western blotting analysis of the cell wall protein fraction
3)

biosynthesis of glycans in the cell wall.
Cell wall proteins were then extracted from strain
DAN8677 and the disruptants of orthologs AN5663 andAN2015. Western blotting analysis of the cell wall protein
fraction using anti-b 1,5-galactofuranose antibody revealed
that b 1,5-galactofuranose residues were not present in thecell wall of the strain DAN8677 (Fig. 2).

4)

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based on amino acid sequence similarities. Biochem. J., 326: 929–939.
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