Appl Microbiol Biotechnol (2010) 87:1783–1789 DOI 10.1007/s00253-010-2664-6 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module Soichiro Tabuchi & Junji Ito & Takashi Adachi & Hiroki Ishida & Yoji Hata & Fumiyoshi Okazaki & Tsutomu Tanaka & Chiaki Ogino & Akihiko Kondo Received: 24 March 2010 / Revised: 27 April 2010 / Accepted: 29 April 2010 / Published online: 25 May 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM- GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein. S. Tabuchi : J. Ito : T. Adachi : C. Ogino : A. Kondo (*) Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan e-mail: akondo@kobe-u.ac.jp H. Ishida : Y. Hata Research Institute, Gekkeikan Sake Co., Ltd, 300 Katahara-cho, Fushimi-ku, Kyoto 612-8361, Japan F. Okazaki : T. Tanaka Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan Keywords Cell surface display . Aspergillus oryzae . Chitin-binding module . Triacylglycerol lipase Introduction The development of cell surface display systems of heterologous proteins or peptides has recently become an active research area (Boder and Wittrup 1997; Stahl and Uhlen 1997; Kondo and Ueda 2004). By utilizing naturally occurring surface proteins as scaffolds, functional proteins can be displayed on various cell surfaces (Hansson et al. 2001). In these systems, cells displaying functional proteins can be easily separated and have been used in a wide variety of biotechnological applications, such as bioconversion (Tateno et al. 2007; Kotaka et al. 2008), bioremediation of heavy metals (Kambe-Honjoh et al. 2000), oral vaccine development (Raha et al. 2005; Ramasamy et al. 2006), and combinatorial library screening (Georgiou et al. 1997). Cell surface display systems require anchor proteins as a scaffold to maintain fused proteins with a high degree of stability within the cell wall. So far, several cell surface display systems using various anchor proteins have been reported in Gram-negative/positive bacteria, yeasts, and so on (Murai et al. 1997; Lee et al. 2003; Tanino et al. 2004). Aspergillus oryzae has been commonly used in the manufacture of traditional Japanese fermented food products, such as sake, miso (paste made from soybeans), and soy sauce, and has been guaranteed to be generally recognized as safe (GRAS). Recently, the molecular biology of Aspergillus species has been studied extensively, including determination of both the entire genome sequence and the expressed sequence tags (Machida et al. 2005; Akao et al. 2007).