Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences

1 Molecular Microbiology and Biotechnology Group, Research Institute of Innovative Technology for the Earth (RITE), 9-2, Kizugawadai, Kizugawa, Kyoto 619-0292, Japan 2 Honda R&D Co., Ltd, 1-4-1 Chuo Wako, Saitama 351-0193, Japan Correspondence Hideaki Yukawa mmg-lab{at}rite.or.jp Systematic screening of secretion proteins using an approach based on the completely sequenced genome of Corynebacterium glutamicum R revealed 405 candidate signal peptides, 108 of which were able to heterologously secrete an active-form -amylase derived from Geobacillus stearothermophilus . These comprised 90 general secretory (Sec)-type, 10 twin-arginine translocator (Tat)-type and eight Sec-type with presumptive lipobox peptides. Only Sec- and Tat-type signals directed high-efficiency secretion. In two assays, 11 of these signals resulted in 50- to 150-fold increased amounts of secreted -amylase compared with the well-known corynebacterial secretory protein PS2. While the presence of an AXA motif at the cleavage sites was readily apparent, it was the presence of a glutamine residue adjacent to the cleavage site that may affect secretion efficiency. Abbreviations: Sec, general secretory; SPase, signal peptidase; Tat, twin-arginine translocator Two supplementary tables, listing oligonucleotide DNA primers used in this study and putative secretory proteins examined in this work, are available with the online version of this paper.