The one-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase

Zhao, Chao; Wu, Yijing; Yu, Hai; Shah, Ishita M.; Li, Yanhong; Zeng, Jie; Liu, Bin; Mills, David A.; Chen, Xi

doi:10.1039/c5cc10646j

Cited by 60 publications

(37 citation statements)

References 41 publications

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“…Yu and Chen developed 12 effective one-pot multienzyme (OPME) systems for the direct production of eight sugar nucleotides, including UDP-Glc, UDP-GlcNAc, UDP-GalNAc, UDP-Gal, UDP-GlcA, GDP-Man, GDP-Fuc, and CMP-Sia, from their corresponding simple monosaccharides (13). Sequential assemblies of those OPME systems have synthesized the lacto-N-neotetraose and its sialyl and fucosyl derivatives in the preparative scale (14,15).…”

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confidence: 99%

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Tian

Yang

Zeng

et al. 2019

Appl Environ Microbiol

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Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained. IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

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confidence: 99%

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Tian

Yang

Zeng

et al. 2019

Appl Environ Microbiol

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“…The α1–2-fucose was installed to type I disaccharide Galβ3GlcNAcβProN 3 ( 1 ) to produce H type I trisaccharide 22 Fucα2Galβ3GlcNAcβProN 3 ( 21 ) by a one-pot three-enzyme fucosylation system containing BfFKP, PmPpA, and a highly efficient α1–2-fucosyltransferase cloned from Thermosynechococcus elongatus (Te2FT). 41 Le b tetrasaccharide Fucα2Galβ3(Fucα4)GlcNAcβProN 3 ( 22 ) was successfully obtained in 98% yield, indicating α1–2-fucosylation of type I glycan did not affect its recognition by Hp3/4FT as an acceptor. However, attempts to alter the fucosylation sequence by performing Te2FT-catalyzed α1–2-fucosylation of Le a ( 11 ) was not successful, indicating that α1–3-fucosylation of type I glycan blocked Te2FT activity.…”

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confidence: 99%

H. pylori α1–3/4-fucosyltransferase (Hp3/4FT)-catalyzed one-pot multienzyme (OPME) synthesis of Lewis antigens and human milk fucosides

et al. 2017

Chem. Commun.

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Helicobacter pylori α1–3/4-fucosyltransferase (Hp3/4FT) was expressed in Escherichia coli at a level of 30 mg/L culture and used as a diverse catalyst in a one-pot multienzyme (OPME) system for high-yield production of L-fucose-containing carbohydrates including Lewis antigens such as Lewis a, b, x, O-sulfated Lewis x, and sialyl Lewis x as well as human milk fucosides such as 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-difuco-hexaose (LNDFH) II and III. Noticeably, while difucosylation of tetrasaccharides was readily achieved using an excess amount of donor, synthesis of LNFP III was achieved by Hp3/4FT-catalyzed selective fucosylation of the N-acetyllactoamine (LacNAc) component in lacto-N-neotetraose (LNnT).

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“…Here, we report a chemoenzymatic strategy that can give easy access to a library of differentially fucosylated and sialylated oligo‐LacNAc derivatives starting from advanced precursor. It is based on the finding that lactosamine derivatives with a free amine or the amino function modified by a tert ‐butyloxycarbonyl (Boc) protecting group, are resistant to fucosylation by recombinant FUT5 and Hp39‐FT (Figure a) . To exploit this finding, we prepared precursor 1 (Figure b), which through simple modifications could be converted into six different hexasaccharides ( 2 – 7, Figure c) in which the glucosamine moieties are either acetylated (GlcNAc) or modified with a free amine (GlcNH 2 ) or Boc (GlcNHBoc).…”

Section: Figurementioning

confidence: 81%

Protecting‐Group‐Controlled Enzymatic Glycosylation of Oligo‐N‐Acetyllactosamine Derivatives

Gagarinov

Wei

et al. 2019

Angew Chem Int Ed

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We describe achemoenzymatic strategy that can give al ibrary of differentially fucosylated and sialylated oligosaccharides starting from as ingle chemically synthesized tri-Nacetyllactosamine derivative.T he common precursor could easily be converted into 6d ifferent hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as af ree amine (GlcNH 2 )o rB oc (GlcNHBoc). Fucosylation of the resulting compounds by ar ecombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties,p roviding access to 6selectively mono-and bis-fucosylated oligosaccharides.Conversion of the GlcNH 2 or GlcNHBoc moieties into the natural GlcNAc,f ollowed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds.T he oligosaccharides were printed as am icroarray that was probed by several glycan-binding proteins,d emonstrating that complex patterns of fucosylation can modulate glycan recognition.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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The one-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase

Cited by 60 publications

References 41 publications

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System

H. pylori α1–3/4-fucosyltransferase (Hp3/4FT)-catalyzed one-pot multienzyme (OPME) synthesis of Lewis antigens and human milk fucosides

Protecting‐Group‐Controlled Enzymatic Glycosylation of Oligo‐N‐Acetyllactosamine Derivatives

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