{"doi":"10.1186/s12934-019-1058-4","title":"Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein","abstract":null,"journal":"Microbial Cell Factories","year":2019,"id":678585,"datarank":0.5456379239589579,"base_score":3.6375861597263857,"endowment":3.6375861597263857,"self_citation_contribution":0.5456379239589579,"citation_network_contribution":0.0,"self_endowment_contribution":0.5456379239589579,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":37,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":716553,"name":"Nitin S. Kamble","orcid":"0000-0001-5521-9737","position":1,"is_corresponding":false},{"id":1772997,"name":"Elizabeth K. Court","orcid":null,"position":2,"is_corresponding":false},{"id":770492,"name":"Owain J. Bryant","orcid":"0000-0003-2683-038X","position":3,"is_corresponding":false},{"id":1717826,"name":"Matthew G. Hicks","orcid":null,"position":4,"is_corresponding":false},{"id":1773001,"name":"Christopher Lennon","orcid":null,"position":5,"is_corresponding":false},{"id":1773002,"name":"Gillian M. Fraser","orcid":null,"position":6,"is_corresponding":false},{"id":723483,"name":"Phillip C. Wright","orcid":"0000-0002-8834-0426","position":7,"is_corresponding":false},{"id":616149,"name":"Graham P. Stafford","orcid":"0000-0002-5600-2465","position":8,"is_corresponding":false},{"id":1772994,"name":"Charlotte A. Green","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein","abstract":"Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the ‘hijack’ of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5′UTR and first 47 amino acidsof FliC from E. coli (but no 3′UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L−1, in low cell density culture. We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.","is_dataset_classified":null,"base_score":3.6375861597263857,"endowment":3.6375861597263857,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"30657054","pmcid":"PMC6337784","openalex_id":"https://openalex.org/W2910784510","authors":[],"funders":[{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"CBMNet Business Innovation Voucher","title":null},{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"BB/J016322/1","title":"Use of Synthetic Biology in the Development of Bacterial Adhesins for Skin Grafting applications"},{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"BB/M007197/1","title":"Sequential assembly of the bacterial flagellum outside the living cell"},{"funder_name":"Department of Pathology, University of Cambridge","grant_id":"John Lucas Walker studentship","title":null},{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"BB/F018681/1","title":null},{"funder_name":"Biotechnology and Biological Sciences Research Council","grant_id":"BB/K011200/1","title":null},{"funder_name":"University of Sheffield","grant_id":"","title":null},{"funder_name":"Government of India","grant_id":"","title":null}],"total_grants":8,"fwci":1.6084,"citation_percentile":0.83379723,"influential_citations":0,"citation_trend":[{"year":2020,"count":6},{"year":2021,"count":2},{"year":2022,"count":2},{"year":2023,"count":3},{"year":2024,"count":4},{"year":2025,"count":9},{"year":2026,"count":11}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-019-1058-4","host_type":"journal"},{"url":"https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-019-1058-4","host_type":"publisher"},{"url":"http://link.springer.com/content/pdf/10.1186/s12934-019-1058-4.pdf","host_type":"publisher"},{"url":"http://link.springer.com/article/10.1186/s12934-019-1058-4/fulltext.html","host_type":"publisher"},{"url":"https://doi.org/10.1186/s12934-019-1058-4","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/30657054","host_type":"repository"},{"url":"https://eprints.whiterose.ac.uk/141412/1/s12934-019-1058-4.pdf","host_type":"repository"},{"url":"http://europepmc.org/pmc/articles/PMC6337784","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/6337784","host_type":"repository"},{"url":"https://doaj.org/article/e452668ad75543f096a42f8f752f7056","host_type":"repository"},{"url":"https://www.repository.cam.ac.uk/handle/1810/288217","host_type":"repository"},{"url":"https://www.repository.cam.ac.uk/handle/1810/289451","host_type":"repository"},{"url":"https://doi.org/10.17863/cam.35533","host_type":"repository"},{"url":"https://doi.org/10.17863/cam.36700","host_type":"repository"},{"url":"https://microbialcellfactories.biomedcentral.com/track/pdf/10.1186/s12934-019-1058-4","host_type":"Unpaywall"},{"url":"https://europepmc.org/articles/PMC6337784","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC6337784?pdf=render","host_type":"Europe_PMC"},{"url":"https://dx.doi.org/10.17863/cam.35533","host_type":""},{"url":"https://dx.doi.org/10.17863/cam.36700","host_type":""},{"url":"http://dx.doi.org/10.1186/s12934-019-1058-4","host_type":""},{"url":"https://dx.doi.org/10.1186/s12934-019-1058-4","host_type":""},{"url":"https://eprints.whiterose.ac.uk/id/eprint/141412/","host_type":""},{"url":"https://doi.org/https://doi.org/10.1186/s12934-019-1058-4","host_type":""}],"fields_of_study":["Bacterial Genetics and Biotechnology","Escherichia coli research studies","Cellular transport and secretion","0301 basic medicine","0303 health sciences","03 medical and health sciences","5' Untranslated Regions","Carboxylic Ester Hydrolases","Escherichia coli","Escherichia coli Proteins","Flagella","Flagellin","Human Growth Hormone","Humans","Metabolic Engineering","Recombinant Proteins","Thioredoxins","Type III Secretion Systems"],"mesh_terms":["Type III Secretion Systems","Carboxylic Ester Hydrolases","Escherichia coli","Flagella","Flagellin","Humans","Recombinant Proteins","Thioredoxins","Human Growth Hormone","5' Untranslated Regions","Escherichia coli Proteins","Metabolic Engineering"],"keywords":["Secretion","Periplasmic space","Heterologous","Flagellum","Secretory protein","Cutinase","Biology","Type VI secretion system","Biochemistry","Secretory pathway","Escherichia coli","Recombinant DNA","Flagellin","Heterologous expression","Cell biology","Enzyme","Endoplasmic reticulum","Gene","Golgi apparatus","Biotechnology","flagellar","Synthetic Biology","Strain Engineering","Recombinant Protein Secretion","Secretion Assay","Human Growth Hormone","Research","Escherichia coli Proteins","Microbiology","QR1-502","Recombinant Proteins","Thioredoxins","Metabolic Engineering","Flagella","Type III Secretion Systems","Humans","5' Untranslated Regions","Carboxylic Ester Hydrolases"],"sdg_mappings":[{"sdg_number":3,"sdg_label":"3. Good health"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[{"name":"gen"}],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-17T05:38:27.913768Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}