Genome-scale metabolic model-based engineering of Escherichia coli enhances recombinant single-chain antibody fragment production. 2022

Aidin Behravan, and Atieh Hashemi, and Sayed-Amir Marashi, and Hamideh Fouladiha
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, ValiAsr Avenue, Niayesh Junction, PO Box 14155-6153, Tehran, Iran.

OBJECTIVE Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a small antibody fragment that has received considerable attention for its ability to target the epithelial cell adhesion molecule (EpCAM), a cancer-associated biomarker of solid tumors. Due to its metabolic burden, scFv recombinant expression causes a remarkable decrease in the maximum specific growth rate of the scFv-producing strain. In the present study, a genome-scale metabolic model (GEM)-guided engineering strategy is proposed to identify gene targets for improved antiEpEX-scFv production in E. coli. METHODS In this study, a genome-scale metabolic model of E. coli (iJO1366) and a metabolic modeling tool (FVSEOF) were employed to find appropriate genes to be amplified in order to improve the strain for incresed production of antiEpEX-scFv. To validate the model predictions, one target gene was overexpressed in the parent strain Escherichia coli BW25113 (DE3). RESULTS For improving scFv production, we applied the FVSEOF method to identify a number of potential genetic engineering targets. These targets were found to be localized in the glucose uptake system and pentose phosphate pathway. From the predicted targets, the glk gene encoding glucokinase was chosen to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). By overexpressing glk, the growth capacity of the recombinant E. coli strain was recovered. Moreover, the engineered strain with glk overexpression successfully led to increased scFv production. CONCLUSIONS The genome-scale metabolic modeling can be considered for the improvement of the production of other recombinant proteins.

UI MeSH Term Description Entries
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005941 Glucokinase A group of enzymes that catalyzes the conversion of ATP and D-glucose to ADP and D-glucose 6-phosphate. They are found in invertebrates and microorganisms, and are highly specific for glucose. (Enzyme Nomenclature, 1992) EC 2.7.1.2.
D005947 Glucose A primary source of energy for living organisms. It is naturally occurring and is found in fruits and other parts of plants in its free state. It is used therapeutically in fluid and nutrient replacement. Dextrose,Anhydrous Dextrose,D-Glucose,Glucose Monohydrate,Glucose, (DL)-Isomer,Glucose, (alpha-D)-Isomer,Glucose, (beta-D)-Isomer,D Glucose,Dextrose, Anhydrous,Monohydrate, Glucose
D000071858 Epithelial Cell Adhesion Molecule A cell adhesion molecule that is expressed on the membranes of nearly all EPITHELIAL CELLS, especially at the junctions between intestinal epithelial cells and intraepithelial LYMPHOCYTES. It also is expressed on the surface of ADENOCARCINOMA and epithelial tumor cells. It may function in the MUCOSA through homophilic interactions to provide a barrier against infection. It also regulates the proliferation and differentiation of EMBRYONIC STEM CELLS. Antigen, CD326,CD326 Protein,ESA Antigen,Ep-CAM,EpCAM,Epithelial Specific Antigen,GA 733 Tumor-Associated Antigen,GA733 Antigen,GA733 Tumor-Associated Antigen,Tacstd1 Protein,Tumor-Associated Antigen GA733,Antigen, ESA,Antigen, Epithelial Specific,Antigen, GA733,CD326 Antigen,GA 733 Tumor Associated Antigen,GA733 Tumor Associated Antigen,GA733, Tumor-Associated Antigen,Tumor Associated Antigen GA733,Tumor-Associated Antigen, GA733
D015415 Biomarkers Measurable and quantifiable biological parameters (e.g., specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health- and physiology-related assessments, such as disease risk, psychiatric disorders, ENVIRONMENTAL EXPOSURE and its effects, disease diagnosis; METABOLIC PROCESSES; SUBSTANCE ABUSE; PREGNANCY; cell line development; EPIDEMIOLOGIC STUDIES; etc. Biochemical Markers,Biological Markers,Biomarker,Clinical Markers,Immunologic Markers,Laboratory Markers,Markers, Biochemical,Markers, Biological,Markers, Clinical,Markers, Immunologic,Markers, Laboratory,Markers, Serum,Markers, Surrogate,Markers, Viral,Serum Markers,Surrogate Markers,Viral Markers,Biochemical Marker,Biologic Marker,Biologic Markers,Clinical Marker,Immune Marker,Immune Markers,Immunologic Marker,Laboratory Marker,Marker, Biochemical,Marker, Biological,Marker, Clinical,Marker, Immunologic,Marker, Laboratory,Marker, Serum,Marker, Surrogate,Serum Marker,Surrogate End Point,Surrogate End Points,Surrogate Endpoint,Surrogate Endpoints,Surrogate Marker,Viral Marker,Biological Marker,End Point, Surrogate,End Points, Surrogate,Endpoint, Surrogate,Endpoints, Surrogate,Marker, Biologic,Marker, Immune,Marker, Viral,Markers, Biologic,Markers, Immune
D057127 Single-Chain Antibodies A form of antibodies consisting only of the variable regions of the heavy and light chains (FV FRAGMENTS), connected by a small linker peptide. They are less immunogenic than complete immunoglobulin and thus have potential therapeutic use. Fv Antibody Fragments, Single-Chain,ScFv Antibodies,Single-Chain Fv,Single-Chain Fv Antibody,Single-Chain Fv Antibody Fragments,Single-Chain Variable Fragment,Single-Chain Variable Fragment Antibodies,Single-Chain Variable Fragment Antibody,Single-Chain Variable Fragments,Antibodies, ScFv,Antibodies, Single-Chain,Antibody, Single-Chain Fv,Fragment, Single-Chain Variable,Fragments, Single-Chain Variable,Fv Antibody Fragments, Single Chain,Fv Antibody, Single-Chain,Fv, Single-Chain,Single Chain Antibodies,Single Chain Fv,Single Chain Fv Antibody,Single Chain Fv Antibody Fragments,Single Chain Variable Fragment,Single Chain Variable Fragment Antibodies,Single Chain Variable Fragment Antibody,Single Chain Variable Fragments,Variable Fragment, Single-Chain,Variable Fragments, Single-Chain
D060847 Metabolic Engineering Methods and techniques used to genetically modify cells' biosynthetic product output and develop conditions for growing the cells as BIOREACTORS. Engineering, Metabolic

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