RESUMO
Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram-scale by high-cell-density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for alpha-3 fucosyltransferase and the Neisseria meningitidis lgtB gene for beta-4 galactosyltransferase. The first compound Galbeta-4(Fucalpha-3)GlcNAcbeta-4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin-synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galbeta-4(Fucalpha-3)GlcNAcbeta-3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for beta-3 N-acetylglucosaminyltransferase.
Assuntos
Escherichia coli/metabolismo , Oligossacarídeos/biossíntese , Trissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Escherichia coli/química , Antígenos CD15/análogos & derivados , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificaçãoRESUMO
Large amounts of globotriose (Galalpha-4Galbeta-4Glc) are shown to be produced by the high cell density culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta galactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose, globotriose could serve as an alternative acceptor for LgtC and the formation of a series of polygalactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAcbeta-3Galalpha-4Galbeta-4Glc) by overexpressing two additional genes: lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase. Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken up by the cells.
Assuntos
Escherichia coli/metabolismo , Globosídeos/biossíntese , Trissacarídeos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Globosídeos/genética , Trissacarídeos/genéticaAssuntos
Escherichia coli/metabolismo , Gangliosídeos/biossíntese , Engenharia Genética/métodos , Oligossacarídeos/biossíntese , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Escherichia coli/genética , Gangliosídeos/química , Inativação Gênica , Lactose/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Ácido N-Acetilneuramínico/metabolismo , N-Acilneuraminato Citidililtransferase/genética , N-Acilneuraminato Citidililtransferase/metabolismo , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Oligossacarídeos/química , Oxo-Ácido-Liases/genética , Oxo-Ácido-Liases/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transformação Bacteriana , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Oligo- and polysaccharides embodying the alpha-maltotriosyl-6(II)-maltotetraosyl structure were readily synthesized by transglycosylation of maltosyl fluoride onto panose and pullulan catalysed by the bacterial transglycosylase cyclodextrin glycosyltransferase (CGTase). The two products obtained proved useful for increasing the knowledge of substrate binding and processing at the active site of barley limit dextrinase that is involved in the metabolism of amylopectin by acting upon its branch points.