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A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A.

Pieretti G, Cipolletti M, D’Alonzo D, Alfano A, Cimini D, Cammarota M, Palumbo G, Giuliano M, De Rosa M, Schiraldi C, Parrilli M, Bedini E, Corsaro MM.

Appl Microbiol Biotechnol. 2014 ;98(18):7781-91.

Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, 80126, Napoli, Italy.

 

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 lipidA 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.

 

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