Cell-Surface Displayed Expression of Trehalose Synthase from Pseudomonas putida ATCC 47054 in Pichia Pastoris Using Pir1p as an Anchor Protein

ABSTRACT The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl2. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.

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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

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.269-275.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 269-275 ◽

Cited By ~ 83

Author(s):

Junya Narita ◽

Kenji Okano ◽

Tomoe Kitao ◽

Saori Ishida ◽

Tomomitsu Sewaki ◽

...

Keyword(s):

Lactic Acid ◽

Cell Surface ◽

Fusion Protein ◽

Lactobacillus Casei ◽

Surface Display ◽

Flow Cytometric Analysis ◽

Soluble Starch ◽

Engineering System ◽

Acid Yield ◽

Anchor Protein

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 α-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.

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Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01525-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6217-6224 ◽

Cited By ~ 99

Author(s):

Thomas Baumgarten ◽

Stefanie Sperling ◽

Jana Seifert ◽

Martin von Bergen ◽

Frank Steiniger ◽

...

Keyword(s):

Stress Response ◽

Cell Surface ◽

Pseudomonas Putida ◽

Biofilm Formation ◽

Membrane Vesicle ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Outer Membrane Vesicles ◽

Stress Factors ◽

Content Type

ABSTRACTAmong the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that ofPseudomonas aeruginosabiofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response ofPseudomonas putidaDOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

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