Functional analysis of SM proteins for heterologous protein
production in Aspergillus oryzae
Yujiro HIGUCHI
Faculty of Agriculture, Kyushu University
Research aims
In eukaryotic cells, secretory proteins are transported to the extracellular space via
vesicles detached from the endoplasmic reticulum (ER), Golgi apparatus, through the plasma
membrane. In this vesicular trafficking pathway, soluble N-ethylmaleimide sensitive factor(NSF) attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins are requiredfor fusion of the vesicle with the target membrane. SNARE proteins localize on target orvesicle membranes, where they form complexes that aid in membrane fusion; SM proteinsact on SNARE protein complex to assist in this function. SM proteins have been wellanalyzed in the model eukaryote Saccharomyces cerevisiae, a type of yeast. In this model,
the protein Sec1p functions from Golgi to the plasma membrane, and the Sly1p proteinfunctions from ER to Golgi. It has been previously reported that S. cerevisiae cells thatoverexpressed SM proteins produced greater amounts of α-amylase derived from Aspergillus
oryzae1. In contrast, in the model filamentous fungus A. oryzae, no information about theeffect of SM proteins has been reported. A. oryzae has been approved as the Japanesenational fungus because it is important in the Japanese fermentation and brewing industry
and because it can produce abundant secretory proteins, including α-amylase. In this study, I
aimed to elucidate the role of SM proteins in A. oryzae that are thought to function in thesecretory pathway, using functional analysis as a basic study and trying to produce valuableproteins as an applied study.
Methods
In the genome database of A. oryzae (http://www.aspgd.org/), we found four predicted
SM proteins, in which two proteins were orthologs of S. cerevisiae secretory SM proteins.
One was an ortholog of Sec1p, named AoSec1 (AO090010000460), and the other was an
ortholog of Sly1p, named AoSly1 (AO090701000606). We determined that the amino acidsequences of both A. oryzae proteins contain a Sec1 domain, suggesting that these A. oryzae
SM proteins have a conserved function. Aosec1 and Aosly1 were cloned into an
egfp-containing vector to generate strains expressing each EGFP-fused protein forlocalization analysis.
In the S. cerevisiae genome database (https://www.yeastgenome.org/), it was reported
that SEC1 and SLY1 are essential genes. Therefore, Aosec1 or Aosly1
conditionally-expressing strains were produced where each gene was regulated by the nmtA
promoter (PnmtA), which has been successfully used for the analysis of essential genes2.
SM protein-overexpressing strains, where the amyB promoter (PamyB) is used to express
each SM protein-encoding gene, were generated to analyze the effect of secretory proteinamount. α-Amylase (AmyB) activity in the culture supernatant of these SM
protein-overexpressing strains was analyzed as a homologous secretory protein using anα-amylase measuring kit (Kikkoman), and total secretory protein amount was determinedusing Bradford dye reagent (Takara).
Results
1. Localization analysis of A. oryzae SM proteins
The AoSec1 or AoSly1 encoding gene was cloned, and strains expressing eachC-terminally EGFP-tagged protein were cultured for observation under a fluorescent
microscope. AoSec1-EGFP was observed to locate in the cytoplasm, while AoSly1-EGFP
was observed to exist as punctate structures, suggesting Golgi localization (Fig. 1).

2. Analysis of conditional mutants of A. oryzae SM proteinsConditional mutants, in which expression of Aosec1 or Aosly1 is regulatable by PnmtA,
were generated, and their growths were analyzed on plates of M (minimal) or PD (rich)medium, supplemented with or without 10 µM thiamine. In Aosec1-repressed conditions, noobvious growth defect was observed. In contrast, in Aosly1-repressed conditions, severe
growth defect was observed (Fig. 2). In S. cerevisiae, SEC1 and SLY1 are essential genes;
however, in A. oryzae, Aosec1 was shown to be non-essential, at least under the examinedconditions.
3. Analysis of protein secretion in strains overexpressing A. oryzae SM proteins
Aosec1- or Aosly1-overexpressing strains werecultured in DPY medium for 2 days, and total
secretory protein amount was measured in theculture supernatant (Fig. 3). SM proteinoverexpressing strains could produce approximately
1.3-fold more secretory protein than a control strain.
However, no increase in α-amylase activity wasobserved. Heterologous protein (bovine chymosin)
production will be examined in the future study.

Conclusion
AoSec1 localizes in the cytoplasm and AoSly1 likely localizes to Golgi apparatus. In
Aosec1-repressed conditions, no obvious growth phenotype was observed; in contrast, in
Aosly1-repressed conditions, a severe growth defect was observed. Both Aosec1- andAosly1-overexpressing strains produced more secretory proteins, suggesting that SM
proteins in A. oryzae function in the secretory pathway. In a future study, the effect of SM
protein-repressed or -overexpressed conditions on heterologous protein production will beexamined.
References
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trafficking improves heterologous protein secretion in Saccharomyces cerevisiae. Metab.
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2) Hiramoto, T., Tanaka, M., Ichikawa, T., Matsuura, Y., Hasegawa-Shiro, S., and Gomi, K.
(2015) Endocytosis of a maltose permease is induced when amylolytic enzyme production
is repressed in Aspergillus oryzae. Fungal Genet. Biol. 82: 136–144.
3) Yoon, J., Maruyama, J., and Kitamoto, K. (2010) Disruption of ten protease genes in thefilamentous fungus Aspergillus oryzae highly improves production of heterologous
proteins. Appl. Microbiol. Biotechnol. 89: 747–759.