Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface ofLactobacillus plantarum, a Potent Member of the Gut Microbiome

ABSTRACTHeterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the “adaptor scaffoldin” strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia ofLactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all producedin vivoby the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCEThe multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering ofLactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

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Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Applied and Environmental Microbiology ◽

10.1128/aem.06127-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 560-567 ◽

Cited By ~ 7

Author(s):

Denise Knobloch ◽

Kai Ostermann ◽

Gerhard Rödel

Keyword(s):

Cell Surface ◽

Bacillus Megaterium ◽

Fusion Proteins ◽

Shuttle Vector ◽

Surface Display ◽

Fluorescent Labeling ◽

Content Type ◽

Sporosarcina Ureae

ABSTRACTMonomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report thatBacillus megateriumis an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA fromSporosarcina ureaeATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into theEscherichia coli-Bacillus megateriumshuttle vector pHIS1525. After transformation of the respective plasmids intoBacillus megateriumprotoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemblein vitrointo highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope ofBacillus megaterium, indicating that the cell surface can servein vivoas a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

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Cover Feature: Bacterial Cell‐Surface Display of Semisynthetic Cyclic Peptides (ChemBioChem 1/2019)

ChemBioChem ◽

10.1002/cbic.201800748 ◽

2018 ◽

Vol 20 (1) ◽

pp. 4-4

Author(s):

Shubhendu Palei ◽

Kira S. Becher ◽

Christian Nienberg ◽

Joachim Jose ◽

Henning D. Mootz

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Cyclic Peptides ◽

Surface Display ◽

Cell Surface Display ◽

Bacterial Cell Surface

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Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety

Applied and Environmental Microbiology ◽

10.1128/aem.02223-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 43-53 ◽

Cited By ~ 18

Author(s):

Joseph P. Park ◽

Min-Jung Choi ◽

Se Hun Kim ◽

Seung Hwan Lee ◽

Haeshin Lee

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Communication Systems ◽

Cell Attachment ◽

Surface Display ◽

Content Type ◽

E Coli ◽

Material Surfaces ◽

Mussel Adhesive ◽

Adhesive Proteins

ABSTRACTMussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine onEscherichia colisurfaces using outer membrane protein W as an anchoring motif for the first time. Localization of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence microscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catecholamine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catecholamines. The engineeredE. coliexhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethylene terephthalate), poly(urethane), and poly(dimethylsiloxane). The uniqueness of this approach utilizing the engineered stickyE. coliis that no chemistry for cell attachment are necessary, and the ability of spontaneousE. coliattachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded stickyE. colithat can be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell communication systems, and many others.

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Discontinuous Epitope Mapping of Antibodies Using Bacterial Cell Surface Display of Folded Domains

Epitope Mapping Protocols - Methods in Molecular Biology ◽

10.1007/978-1-4939-7841-0_11 ◽

2018 ◽

pp. 159-183

Author(s):

Anna-Luisa Volk ◽

Johan Rockberg

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Epitope Mapping ◽

Surface Display ◽

Cell Surface Display ◽

Discontinuous Epitope ◽

Bacterial Cell Surface

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Impact of Resolvin E1 on Murine Neutrophil Phagocytosis in Type 2 Diabetes

Infection and Immunity ◽

10.1128/iai.02444-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 792-801 ◽

Cited By ~ 46

Author(s):

Bruno S. Herrera ◽

Hatice Hasturk ◽

Alpdogan Kantarci ◽

Marcelo O. Freire ◽

Olivia Nguyen ◽

...

Keyword(s):

Type 2 Diabetes ◽

Transgenic Animals ◽

Genetically Engineered ◽

Mitogen Activated Protein Kinase ◽

Air Pouch ◽

Content Type ◽

Genetically Engineered Mice ◽

The Impact

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain ofdb/ERV1mice, were used to determine the impact of RvE1 on the phagocytosis ofPorphyromonas gingivalisin T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeledP. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate thein vivoimpact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis ofP. gingivalisin WT animals but had no impact indb/dbanimals. InERV1-transgenic andERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals.In vivodorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis ofP. gingivalisin the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressingERV1.

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Proton-Dependent Gating and Proton Uptake by Wzx Support O-Antigen-Subunit Antiport Across the Bacterial Inner Membrane

mBio ◽

10.1128/mbio.00678-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 29

Author(s):

Salim T. Islam ◽

Paul D. W. Eckford ◽

Michelle L. Jones ◽

Timothy Nugent ◽

Christine E. Bear ◽

...

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Electrical Potential ◽

Three Dimensional ◽

Structural Data ◽

Inner Membrane ◽

Content Type ◽

Bacterial Cell Surface ◽

Repeat Units ◽

Dependent Transport

ABSTRACTWzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx fromPseudomonas aeruginosaPAO1 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPafollowing its purification and reconstitution in phospholipid liposomes. WzxPawas capable of mediating anion flux, consistent with its cationic interior. This flux was electrogenic and modified by extraliposomal pH. Mutation of the above-mentioned acidic residues (E61, D269, and D359) reduced proton (H+)-modified anion flux, showing the role of these amino acid side chains in H+-dependent transport. Wzx also mediated acidification of the proteoliposome interior in the presence of an outward anion gradient. These results indicate H+-dependent gating and H+uptake by WzxPaand allow for the first H+-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM.IMPORTANCEMany bacterial cell surface polysaccharides that are important for survival and virulence are synthesized at the periplasmic leaflet of the inner membrane (IM) using precursors produced in the cytoplasm. Wzx flippases are responsible for translocation of lipid-linked sugar repeat units across the IM and had been previously suggested to simply facilitate passive substrate diffusion. Through our characterization of purified Wzx in a reconstitution system described herein, we have observed protein-dependent intrinsic transport producing a change in the electrical potential of the system, with H+identified as the coupling ion. These results provide the first evidence for coupled (i.e., secondary active) transport by these proteins and, in conjunction with structural data, allow for an antiport mechanism to be proposed for the directed transport of lipid-linked sugar substrates across the IM. These findings bring our understanding of lipid-linked polysaccharide transporter proteins more in line with the efflux pumps to which they are evolutionarily related.

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Bacterial Cell Surface Display

ACS Symposium Series - Next Generation Biomanufacturing Technologies ◽

10.1021/bk-2019-1329.ch005 ◽

2019 ◽

pp. 81-108

Author(s):

Shabnam Parwin ◽

Sashi Kalan ◽

Preeti Srivastava

Keyword(s):

Cell Surface ◽

Bacterial Cell ◽

Surface Display ◽

Cell Surface Display ◽

Bacterial Cell Surface

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Cell Surface Expression of Bacterial Esterase A by Saccharomyces cerevisiae and Its Enhancement by Constitutive Activation of the Cellular Unfolded Protein Response

Applied and Environmental Microbiology ◽

10.1128/aem.00503-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7140-7147 ◽

Cited By ~ 24

Author(s):

Frank Breinig ◽

Björn Diehl ◽

Sabrina Rau ◽

Christian Zimmer ◽

Helmut Schwab ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Surface ◽

Surface Display ◽

Cell Surface Display ◽

Constitutive Activation ◽

Unfolded Protein ◽

Yeast Cell Surface ◽

Protein Response

ABSTRACT Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

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