ABSTRACT
We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying -amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.
Received 29 May 2005/Accepted 28 September 2005
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2006, p. 269–275 Vol. 72, No. 1
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Junya Narita,1 Kenji Okano,2 Tomoe Kitao,2 Saori Ishida,2 Tomomitsu Sewaki,3
Moon-Hee Sung,4 Hideki Fukuda,1 and Akihiko Kondo2*
REVIEW:
The anchoring of proteins to the cell surfaces of bacteria is potentially important in several areas of biotechnological application, including the construction of oral live vaccines and
the production of recombinant proteins. Many related studies have reported the cell surface display of peptides and enzymatically active heterologous proteins in Escherichia coli, Lactobacillus, and Lactococcus. Research into the cell surface display of lactic acid bacteria is aimed mainly at its use as an oral vaccine vehicle (3, 17, 20, 29). Most of the reported cell surface display systems have made use of the C-terminal cell wall-anchoring LPXTG motif (26, 31). This is because many cell surface proteins of gram-positive bacteria have an anchoring region that consists of an LPXTG motif followed by a hydrophobic domain and a charged tail at the predicted C terminus (23). The proteins with the LPXTG motif are cleaved after translocation of the plasma membrane and are amide
linked to a free amino group of the peptide cross-bridge in the cell wall by a postulated sortase (18). Other reported types of display system have made use of S-layer subunits, BspA anchor protein, and AcmA anchor protein. S-layer subunits and BspA are anchored via charge interactions and can be extracted from the cell surface with charge occupying agents such as lithium chloride. The AcmA anchor protein binds specifically to peptidoglycan.In most of the previously reported cell surface display systems mentioned above, the target proteins are displayed by N-terminal fusion to the anchoring motif. In the present study, we developed a new system of cell surface display in lactic acid bacteria by using as an anchor protein Bacillus subtilis subsp.chungkookjang PgsA, which is able to fuse the target protein to its C terminus. PgsA is a transmembrane protein and is one of the poly--glutamate synthetase complexes (1). According to Ashiuchi et al. (2), PgsA functions to stabilize the complex by anchoring in the cell membrane and may function as a poly-glutamate transporter. The PgsA protein is expected to be located between the cell surface and membrane and contains only a transmembrane region consisting of amino acids 25 to 44 as analyzed by the SOSUI (11) system. The PgsA anchor protein is classified as an A1 type anchor (transmembrane anchor) in the system proposed by Leenhouts et al. (16). Since the transmembrane region is located at the N terminus of PgsA, it is predicted that the C terminus of PgsA is exposed outside the cell wall. This is the first report on a cell surface display system of the Lactobacillus genus that uses a transmem- brane protein. For the heterologous target protein, we selected the -amy- lase (AmyA) (EC 3.2.1.1) of the Streptococcus bovis 148 strain, which has a strong ability to hydrolyze and be adsorbed onto corn starch (19, 22). AmyA has been reported to maintain its activity by fusing its N terminus to an anchor protein (24). For the host strain, we selected Lactobacillus casei BLSJ 03135, because it produces 0.8 g of L-lactic acid per g of glucose consumed. We propose to develop a process for direct lactic acid fermentation from starch in which the recombinant lactic acid bacteria with cell surface display of AmyA makes it pos- sible to saccharify starch near the cell surface and utilize the resulting sugar to produce lactic acid.
Corresponding author. Mailing address: Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1
Rokkodaicho, Nada-ku, Kobe 657-8501, Japan. Phone: 81-78-803-6196.
Fax: 81-78-803-6206. E-mail: akondo@kobe-u.ac.jp.
