Przemyslaw Kowal

Reassembled Biosynthetic Pathway for Large‐Scale Carbohydrate Synthesis: α‐Gal Epitope Producing “Superbug”

A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galα1,3Lac through galactose metabolism has been developed. The plasmid contains a λ promoter, a cI857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine–Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galα1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 g L−1) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5′‐diphosphoglucose. This work demonstrates that large‐scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.

Efficient chemoenzymatic synthesis of globotriose and its derivatives with a recombinant α-(1→4)-galactosyltransferase

A truncated alpha-(1-->4)-galactosyltransferase (LgtC) gene from Neisseria meningitidis was cloned. The recombinant glycosyltransferase was expressed in Escherichia coli BL21 (DE3) strain with high specific activity (5 units/mg protein). Its acceptor specificity was carefully characterized. Then the purified enzyme was utilized in highly efficient syntheses of globotriose and a variety of alpha-(1-->4)-galactosylated derivatives as potential antibacterial agents.

Efficient synthesis of globoside and isogloboside tetrasaccharides by using β(1→3) N-acetylgalactosaminyltransferase/ UDP-N-acetylglucosamine C4 epimerase fusion protein

The beta(1-->3) N-acetylgalactosaminyltransferase/UDP-N-acetylglucosamine C4 epimerase fusion protein was constructed and used in coupled enzymatic reactions to synthesize a variety of globotetraose and isoglobotetraose derivatives from the corresponding lactoside acceptors.

Overexpression and biochemical characterization of β-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd

The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K(m) for UDP-GalNAc and globotriose are 58 microM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

ABSTRACT Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcβ1→3Galα1→4Galβ1→4Glc) and isoglobotetraose (GalNAcβ1→3Galα1→3Galβ1→4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant β-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

Synthesis of galactose-containing oligosaccharides through superbeads and superbug approaches: Substrate recognition along different biosynthetic pathways

Publisher Summary Galactosides as carbohydrate receptors play critical roles in biological recognition events. α -galactosyl ( α -gal) epitopes and globotriose are two representative galactosyloligosaccharides with therapeutic significance. Neutralization of anti-gal with free α -gal and its derivatives is a promising treatment to overcome hyper-acute rejection (HAR). It is clear that further studies on preventing HAR and protecting humans from pathogen attack require easy access to substantial amounts of α -gal and globotriose, as well as their analogs. The superbeads and superbug are versatile tools that have shown utility in synthesis of galactoside analogs. At present, other sugar–nucleotide regeneration systems are being constructed to synthesize diverse glycoconjugates and unnatural derivatives. The success of superbeads and the superbug technology offers the promise of easily synthesizing diversified oligosaccharides and glycoconjugates with uncommon or even unnatural sugar residues to meet increasing biochemical demands. On the other hand, the substrate recognition between substrates and enzymes has a dual function. With the superbeads and superbug approaches, it holds promise for the screening of possible inhibitors in the biosynthetic pathway, if the substrates cannot be adopted as suitable donor or acceptors.

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-3H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibodys ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004.