Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

Benkoulouche, Mounir; Fauré, Régis; Remaud‐Siméon, Magali; Moulis, Claire; André, Isabelle

doi:10.1098/rsfs.2018.0069

Cited by 38 publications

(31 citation statements)

References 159 publications

(183 reference statements)

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“…For decades, enzymes have been recognized as useful tools to tackle the complexity of carbohydrate chemistry and surmount the lack of selectivity of chemical catalysts. Among enzyme candidates, Leloir and non-Leloir carbohydrate-active enzymes are widely studied for their ability to synthesize various target glycoconjugates [3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

et al. 2021

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Section: Introductionmentioning

confidence: 99%

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

et al. 2021

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“…Conversely, chemical methods to generate LacNAc require multiple reactive hydroxyl groups and laborious protocols for group protection and deprotection to control the stero- and regio-specificities ( 39 , 40 ). Compared to enzymatic synthesis, generation of LacNAc by chemical synthesis has low yields, a cost-competitive disadvantage for industrial production, hindering the use of LacNAc as an additive in food products ( 30 , 41 , 42 ).…”

Section: Introductionmentioning

confidence: 99%

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome

et al. 2021

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Complex dietary carbohydrate structures including β(1–4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galβ1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound β-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the β-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

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“…Some of them are rather versatile biocatalysts often displaying naturally a relaxed substrate specificity. This promiscuity can be further exacerbated by enzyme engineering to either broaden or narrow down the range of recognized substrates and/or control the reaction selectivity 1 . In particular, mutagenesis targeting the enzyme active site has been very efficient to alter substrate specificity and expand the structural diversity of the accessible glyco-products.…”

Section: Introductionmentioning

confidence: 99%

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

Benkoulouche¹,

Imeddourene²,

Barel³

et al. 2021

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The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

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Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

Cited by 38 publications

References 159 publications

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

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