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Applied and Environmental Microbiology, May 2006, p. 3716-3723, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3716-3723.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Extracellular Production of Neoculin, a Sweet-Tasting Heterodimeric Protein with Taste-Modifying Activity, by Aspergillus oryzae

Ken-ichiro Nakajima,¹ Tomiko Asakura,² Jun-ichi Maruyama,³ Yuji Morita,¹ Hideaki Oike,¹ Akiko Shimizu-Ibuka,^1, Takumi Misaka,¹ Hiroyuki Sorimachi,^1, Soichi Arai,⁴ Katsuhiko Kitamoto,³ and Keiko Abe^1*

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,¹ Laboratory of Food Science, Atomi Junior College, Bunkyo-ku, Tokyo 112-8687, Japan,² Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,³ and Department of Nutritional Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan⁴

Received 17 January 2006/ Accepted 13 March 2006

Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, -amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Department of Nutritional Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.

Present address: Department of Enzymatic Regulation of Cell Functions, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo Metropolitan Organization for Medical Research, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.

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