title: A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A.
abstract: Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, 80126, Napoli, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Pieretti
authors.forename: Manuela
authors.initials: M
authors.lastname: Cipolletti
authors.forename: Daniele
authors.initials: D
authors.lastname: D'Alonzo
authors.forename: Alberto
authors.initials: A
authors.lastname: Alfano
authors.forename: Donatella
authors.initials: D
authors.lastname: Cimini
authors.forename: Marcella
authors.initials: M
authors.lastname: Cammarota
authors.forename: Giovanni
authors.initials: G
authors.lastname: Palumbo
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Giuliano
authors.forename: Mario
authors.initials: M
authors.lastname: De Rosa
authors.forename: Chiara
authors.initials: C
authors.lastname: Schiraldi
authors.forename: Michelangelo
authors.initials: M
authors.lastname: Parrilli
authors.forename: Emiliano
authors.initials: E
authors.lastname: Bedini
authors.forename: Maria Michela
authors.initials: MM
authors.lastname: Corsaro
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D008050:Lipid A / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000276:immunology
chemical_list: D016207:Cytokines; D008050:Lipid A; C048436:monophosphoryl lipid A