Identification of transcription factors and their DNA binding
sequences controlling acid protease production
Youhei YAMAGATA
Graduate School of Agriculture, Tokyo University of Agriculture and Technology
Research aims
Aspergillus oryzae is known as a fungus that produces many types of proteases in large
quantities. However, the mechanism by which the fungi controls production of proteolyticenzymes remains unclear. The protease additives prepared from culture broths of A. oryzae
contain many types of proteases, so it is difficult to make them specific for digestion of theproteins used in industry. We hypothesized that the reason for the difficulty in control ofprotease production was related to nitrogen metabolism in the fungi. Subsequently, we foundthat the nitrogen source in the culture broth affects the production of some proteases 1). In
this study, we analyzed the transcriptional control of the acid protease gene in A. oryzae.
Methods
1) Exploration of the cis-element of the A. oryzae pepO gene and identification of thecorresponding transcriptional factor.
PepO is one of the major proteases in A. oryzae. The DNA sequences 2,024, 1473,
1400, 1300, 1200, 1100, 1000, and 500 bp upstream of pepO were ligated to the amyB
gene and introduced into the A. oryzae triple amylase gene deletion mutant strain,∆amyABC. The transformants were cultured and the amylase activities in the culture
broth were assayed. It was observed that the transcription factor bound to the region-1,200 to -1,100 upstream of the pepO gene. Furthermore, an electrophoretic mobility
shift assay (EMSA) was performed to determine the DNA binding sequence of theputative transcription factor; we identified a 20-bp sequence in the region that wascorrelated with binding.
2) Analysis of the A. oryzae prtR deletion mutant, ∆prtR.
PrtT is found in A. niger and A. fumigatus and is known to comprehensively control
the production of proteases. We identified the homologous gene, prtR, in A. oryzae andconstructed the A. oryzae ΔprtR strain by displacing prtR with ptrA using the A. oryzae
NS4 strain as a host. The deletion strain was cultivated in chemically defined medium
containing casein or Glu and Met and then the acid protease activity was assayed withcasein as a substrate. Additionally, the transcription level of pepO was estimated bysemi-quantitative RT-PCR.

3) Control of acid protease expression with amino acids as a nitrogen source.
From the results above [2)] and our previous study, it was hypothesized that someamino acids in the medium could change the production of acid protease. A. oryzae wascultivated with sodium nitrate as the nitrogen source, and then the mycelia weretransferred new medium containing each amino acid as a solo nitrogen source. After fourhours, the transcription of acid protease genes was estimated by qPCR.
Results1) As the result of a promoter assay using α-amylase, the transcription factor binding site
was identified in the DNA region -1,200 to -1,100 bp upstream of the pepO gene. Then,the binding region was narrowed to 22 bp with EMSA. Two transcription factors werepresumed to be candidates by their DNA binding motif. However, as the DNA-protein
interaction was not inhibited by a 3-bp substitution in their motifs, we hypothesized that
there might be unknown DNA binding proteins, other than the two prospective
transcription factors.
2) The acid protease activity of the ΔprtR strain in the 36-h culture broth was drasticallylower than that of the control strain, and the transcriptional analysis showed pepO wasscarcely transcribed in the ΔprtR strain. The 48-h culture broth showed higher acidprotease activity than that of the 36-h broth, but transcription of pepO was again barely
observed. The change in acid protease activity was particular when cultivating in culture
broth containing Glu. We hypothesized that the transcription of pepO was activated byprtR and that any acid protease other than PepO was expressed at 48 h instead of PepO.
Furthermore, it was shown that Glu up-regulated the acid protease coming up at 48 h.
However, since Met and Glu or sodium nitrite were necessary to cultivate the prtR
deletion mutant, it would be difficult to clarify the reason for the change in activity,
whether it was due to the addition of amino acid or the deletion of the gene. So, weconstructed new prtR deletion mutants from A. oryzae RIB40, ΔligD and ΔpyrG. We arecurrently analyzing these mutants.
3) It was shown that the translation of pepO was activated by Gln, Phe, Val, Leu, Ile, Met,His, and Lys, and that Glu, Asp and Arg suppressed the translation of pepO.
Conclusion
It was shown that the cis-element of the pepO gene, which consisted of less than 22 bp,
was around 1,000 bp upstream of the gene. There were two known motif sequences thatwere recognized by transcription factors, but it was presumed that an unknown protein mightbind to the region, so we have been searching for that protein. It was observed that thecomprehensive transcriptional factor, prtR, in A. oryzae also activated the transcription of

pepO. We are analyzing the new ΔprtR strain in detail. Furthermore, it was shown that someamino acids up-regulated the transcription of pepO, while others suppressed its transcription.
It is hypothesized that these results are related to each other, so we are trying to determine
the mechanism of the control of acid protease production, including PepO.
References
1) Maeda H, Sakai D, Kobayashi T, Morita H, Okamoto A, Takeuchi M, Kusumoto K-I,
Amano H, Ishida H, Yamagata Y (2016) Three extracellular dipeptidyl peptidases foundin Aspergillus oryzae show varying substrate specificities. Appl. Microbiol. Biotechnol.,
100: 4947-4958.