期刊
CARBOHYDRATE RESEARCH
卷 360, 期 -, 页码 19-24出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.carres.2012.07.013
关键词
Glycosaminoglycans; E. coli; K5 polysaccharide; Heparosan; Lyase; Metabolic engineering
资金
- Agence Nationale de la Recherche [ANR-08-BIOT-010-01]
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 beta-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. (C) 2012 Elsevier Ltd. All rights reserved.
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