Large‐Scale Synthesis of H‐Antigen Oligosaccharides by Expressing Helicobacter pylori α1,2‐Fucosyltransferase in Metabolically Engineered Escherichia coli Cells

Abstract

Sugar production: A microbiological method has been developed for the production of fucosyl α1,2‐linked carbohydrates from lactose. The syntheses of 2′‐fucosyllactose and lacto‐N‐neofucopentaose‐1 (see structure), which contain the H‐2 antigen (Fucα‐2Galβ‐4R), were achieved by cultivating Escherichia coli strains that overexpressed the appropriate heterologous genes.

Characterization of Helicobacter pylori α1,2‐Fucosyltransferase for Enzymatic Synthesis of Tumor‐Associated Antigens

“…[17] In contrast, other studies have not been able to reproduce this effect. [8] Thus, in this study the enzyme was generated in stand-alone form and exhibited a specific activity comparable to the reported protein fusion. [17] Although the enzyme has been demonstrated to be soluble, [13] the expression level and specific activity found in this study were much lower compared to the values published for the a1,3- [14] and a1,4-counterparts.…”

“…[21] It has been reported that disrupted futC from strain 26695 can be restored utilizing gene splicing by overlap extension for the modification of the homopolymeric tract. [8] Similarly, futC cloned from strain NCTC11639 was successfully altered in this work by a single step of site-directed mutagenesis after plasmid integration.…”

“…[12,18] A 12-nuleotide sequence was substituted for this tract according to an established approach [8] that was modified by using site-directed mutagenesis to restore the reading frame. Following this procedure, the recombinant protein was successfully overexpressed as a C-terminal His 6 -tagged fusion in E. coli BL21 (DE3) cells and was purified to approximate homogeneity by single-step Ni 2+ affinity chromatography, as confirmed by SDS-PAGE (see Supporting Information, Figure S2).…”

“…[13] Slight variations of this bias could be observed for certain H. pylori source strains. [16] H. pylori a1,2-FucT has been employed for the synthesis of 2'-fucosyllactose [17] and H-type 2 [8] products in vitro and in vivo, respectively, without gaining further information about its selectivity and potency.…”

“…[7] Fermentative procedures are not easily applicable, as these require the time-consuming engineering of the producing or-ganism. [8] Therefore, the specificity and efficiency of enzymatic or chemoenzymatic alternatives likely harbor the highest potential for optimizing the largescale production of fucosylated glycans. [9] Fucosylation, often representing the terminal steps in the biosynthesis of functional carbohydrates, is catalyzed by fucosyltransferases (FucTs).…”