title: Chaperone-assisted expression of KfiC glucuronyltransferase from Escherichia coli K5 leads to heparosan production in Escherichia coli BL21 in absence of the stabilisator KfiB.
abstract: The heparosan synthase of Escherichia coli K5 is composed of the glycosyltransferases KfiA and KfiC which synthesize the polysaccharide heparosan (N-acetylheparosan). A third protein, KfiB, is required to stabilize the KfiAC complex in the bacteria and to transport this complex to the inner membrane where the initiation of polymerization occurs. In this report, we fused KfiC with the E. coli trigger factor (TF) to stabilize KfiC, thus activating the enzyme in the absence of KfiB. Different recombinant plasmids were constructed to compare the impact of the presence or absence of KfiB and the presence of the trigger factor as a fusion protein. Several E. coli BL21-derived strains were transformed with recombinant plasmids and cultivated in fed-batch conditions on minimal medium. The bTCA strain overexpressing fused TF-KfiC together with KfiA and KfiD, but lacking KfiB produced 1.5 g/L of total heparosan after 24 h of fed-batch cultivation. This heparosan was essentially intracellular early in the culture, providing evidence that KfiB primarily plays a role in the exportation process. However, over time, heparosan became mostly extracellular, likely due to passive diffusion or partial cell disruption upon product accumulation.
authors.affiliation: Univ. Grenoble Alpes, CERMAV, F-38000, Grenoble, France.|CNRS, CERMAV, F-38000, Grenoble, France.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Leroux
authors.affiliation: Univ. Grenoble Alpes, CERMAV, F-38000, Grenoble, France. Bernard.Priem@cermav.cnrs.fr.|CNRS, CERMAV, F-38000, Grenoble, France. Bernard.Priem@cermav.cnrs.fr.
authors.forename: Bernard
authors.initials: B
authors.lastname: Priem
mesh_terms: D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015870:Gene Expression; D016695:Glycosyltransferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004187:Disaccharides; D029968:Escherichia coli Proteins; C000613611:KfiB protein, E coli; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C560363:heparosan; D016695:Glycosyltransferases; C097521:KfiC protein, E coli
keywords: Bacterial engineering; Heparosan; Trigger factor