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Applied and Environmental Microbiology, January 2006, p. 269-275, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.269-275.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Display of {alpha}-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

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*

Division of Molecular Science, Graduate School of Science and Technology,1 Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Kobe 657-8501, Japan,2 BioLeaders Japan Corporation, Saito Asagi, Ibaraki 567-0085, Japan,3 Department of Bio- & Nanochemistry, Kookmin University, Songbuk-gu, Seoul 136-702, Korea4

Received 29 May 2005/ Accepted 28 September 2005

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 {alpha}-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.

* 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{at}kobe-u.ac.jp

Applied and Environmental Microbiology, January 2006, p. 269-275, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.269-275.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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