sRNA-Based Screening Chromosomal Gene Targets and Modular Designing Escherichia coli for High-Titer Production of Aglycosylated Immunoglobulin G

AbstractTo construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.

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Estimation of protective levels of anti-O-specific lipopolysaccharide immunoglobulin G antibody against experimental Escherichia coli infection.

Infection and Immunity ◽

10.1128/iai.61.3.975-980.1993 ◽

1993 ◽

Vol 61 (3) ◽

pp. 975-980 ◽

Cited By ~ 17

Author(s):

D E Schiff ◽

C A Wass ◽

S J Cryz ◽

A S Cross ◽

K S Kim

Keyword(s):

Escherichia Coli ◽

Immunoglobulin G ◽

Escherichia Coli Infection

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Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000509708 ◽

2019 ◽

Vol 29 (1-6) ◽

pp. 91-100

Author(s):

Dorna Khoobbakht ◽

Shohreh Zare Karizi ◽

Mohammad Javad Motamedi ◽

Rouhollah Kazemi ◽

Pooneh Roghanian ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Subunit Vaccine ◽

Fusion Gene ◽

Intestinal Epithelial Cells ◽

High Titer ◽

Chimeric Protein ◽

Amino Acid Sequences ◽

Intestinal Epithelial ◽

E Coli

Enterotoxigenic Escherichia coli (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing cooD and cotD genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of E. coli in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in E. coliBL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

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Iha: a Novel Escherichia coli O157:H7 Adherence-Conferring Molecule Encoded on a Recently Acquired Chromosomal Island of Conserved Structure

Infection and Immunity ◽

10.1128/iai.68.3.1400-1407.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1400-1407 ◽

Cited By ~ 229

Author(s):

Phillip I. Tarr ◽

Sima S. Bilge ◽

James C. Vary ◽

Srdjan Jelacic ◽

Rebecca L. Habeeb ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Vibrio Cholerae ◽

Shiga Toxin ◽

Escherichia Coli O157 ◽

Chromosomal Gene ◽

Gene Encoding ◽

Tellurite Resistance ◽

E Coli ◽

Dna Library

ABSTRACT The mechanisms used by Shiga toxin (Stx)-producingEscherichia coli to adhere to epithelial cells are incompletely understood. Two cosmids from an E. coliO157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) ofVibrio cholerae (M. B. Goldberg, S. A. Boyko, J. R. Butterton, J. A. Stoebner, S. M. Payne, and S. B. Calderwood, Mol. Microbiol. 6:2407–2418, 1992). We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha. Iha is 67 kDa in E. coliO157:H7 and 78 kDa in laboratory E. coli and is structurally unlike other known adhesins. DNA adjacent toiha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E. coli, but it is absent from nontoxigenic E. coli O55:H7, sorbitol-fermenting Stx-producing E. coli O157:H−, and laboratory E. coli. We have termed this region the tellurite resistance- and adherence-conferring island. We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E. coli chromosomal island of conserved structure. Pathogenic E. coli O157:H7 has only recently acquired this island.

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Verotoxin-Producing Escherichia coli in Spain: Prevalence, Serotypes, and Virulence Genes of O157:H7 and Non-O157 VTEC in Ruminants, Raw Beef Products, and Humans

Experimental Biology and Medicine ◽

10.1177/153537020322800403 ◽

2003 ◽

Vol 228 (4) ◽

pp. 345-351 ◽

Cited By ~ 71

Author(s):

Jorge Blanco ◽

Miguel Blanco ◽

Jesus E. Blanco ◽

Azucena Mora ◽

Enrique A. Gonzalez ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factor ◽

Intestinal Mucosa ◽

Virulence Genes ◽

Chromosomal Gene ◽

E Coli ◽

Beef Products

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H– [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H–, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H–, O6:H10, O91:H–, O117:H–, O128:H–, O128:H2, O146:H8, O146:H21, O156:H–, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.

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Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.611900 ◽

2020 ◽

Vol 8 ◽

Author(s):

Zhijian Ni ◽

Zhongkui Li ◽

Jinyong Wu ◽

Yuanfei Ge ◽

Yingxue Liao ◽

...

Keyword(s):

Escherichia Coli ◽

Batch Fermentation ◽

Infant Nutrition ◽

High Titer ◽

Production Performance ◽

Cell Factory ◽

Efficient Production ◽

Healthy Development ◽

Fucosyltransferase Gene ◽

Fed Batch Fermentation

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

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Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification

Applied and Environmental Microbiology ◽

10.1128/aem.01014-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7394-7397 ◽

Cited By ~ 47

Author(s):

Jane A. Brockelbank ◽

Verena Peters ◽

Bernd H. A. Rehm

Keyword(s):

Escherichia Coli ◽

Immunoglobulin G ◽

Binding Capacity ◽

Protein A ◽

Coli Strain ◽

Pha Synthase ◽

Enzyme Linked Immunosorbent Assay ◽

Ionization Time ◽

Flight Mass Spectrometry ◽

Igg Binding

ABSTRACT The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.

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