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

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.

Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

“…Second, the displayed molecules are distributed along the lateral cell wall and not concentrated at the septa. This is a distinct advantage compared to other display systems that are localized at the septa (43). Display of proteins along the lateral cell wall should allow optimal exposure of the displayed enzymes toward the substrate.…”

Efficient Production of Optically Pure d -Lactic Acid from Raw Corn Starch by Using a Genetically Modified l -Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

“…This makes the whole process less economically viable. Therefore, many researchers have examined the direct production of lactic acid from starchy materials by using wild amylolytic LAB (ALAB) (6,24,25) or genetically modified amylase-producing LAB (15,16). Although D-lactic acid has been produced by fermentation from pretreated substrates such as rice starch (5) and by simultaneous saccharification and fermentation from cellulose (23), there have been no reports on the direct production of D-lactic acid from starchy materials.…”

Mucosal and Systemic Immune Responses Induced by Recombinant Lactobacillus spp. Expressing the Hemagglutinin of the Avian Influenza Virus H5N1

“…To examine HA 1 expression, the recombinant LA4356-pH and DLD17-pH lactobacilli were grown in MRS broth supplemented with 5 g/ml EM, and cell fractionations and protein extractions were performed as previously described (17 rabbit anti-chicken IgY (1:5,000) conjugated to horseradish peroxidase (HRP) (Sigma) were used. Finally, the blots were developed by enhanced chemiluminescence (ECL) using an ECL Plus detection kit (Thermo Scientific).…”