title: Production of intracellular heparosan and derived oligosaccharides by lyase expression in metabolically engineered E. coli K-12.
abstract: The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding β-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.
authors.affiliation: Centre de Recherche sur les Macromolécules Végétales, Groupe Chimie et Biotechnologie des Oligosaccharides, 601 rue de la Chimie, BP 53X, 38041 Grenoble, Cedex 09, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Barreteau
authors.forename: Emeline
authors.initials: E
authors.lastname: Richard
authors.forename: Sophie
authors.initials: S
authors.lastname: Drouillard
authors.forename: Eric
authors.initials: E
authors.lastname: Samain
authors.forename: Bernard
authors.initials: B
authors.lastname: Priem
mesh_terms: D004187:Disaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D008190:Lyases / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D004187:Disaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C560363:heparosan; D008190:Lyases