Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification

Liang, Chaoning; Zhang, Yi; Jia, Yan; Wang, Wenzhao; Li, Youhai; Lu, Shikun; Jin, Jian‐Ming; Tang, Shuang-Yan

doi:10.1038/srep21051

Cited by 19 publications

(18 citation statements)

References 62 publications

(82 reference statements)

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“…Glycosylation reactions catalyzed by glucansucrases suffer from low thermodynamic favorability as pointed out by Liang et al ( 2016 ). The production of high catechol-G1 concentrations therefore requires an excess of donor substrate sucrose to drive the reaction.…”

Section: Resultsmentioning

confidence: 99%

“…The model acceptor substrate catechol was almost completely glycosylated into monoglycosylated product by the L981A mutant (93 % compared to 60 % for the wild-type enzyme), substantially higher than previously reported for catechol glycosylation by GtfD from S. mutans (65 %) (Meulenbeld and Hartmans 2000 ). In comparison, the I228A Np AS mutant only displayed a luteolin monoglycosylation yield of 53 % (Malbert et al 2014 ), whereas the GtfD mutant showed a catechin monoglycosylation yield of 90 % (Liang et al 2016 ).…”

Section: Discussionmentioning

confidence: 99%

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Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

Devlamynck

Poele

Meng

et al. 2016

Appl Microbiol Biotechnol

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Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of α-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing α-glucan synthesis by mutational engineering of the Gtf180-ΔN enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-ΔN mutants (L938F, L981A, and N1029M) with an impaired α-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-α-d-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-ΔN protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired α-glucan synthesis, thus yielding mutants with an improved glycosylation potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7476-x) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

Devlamynck

Poele

Meng

et al. 2016

Appl Microbiol Biotechnol

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“…Site-saturation mutagenesis targeting the subsite +1 of GTF-D, Tyr418 and Asn469 (equivalent to Tyr430 and Asn481 of GTF180ΔN) was carried out and the mutant library was screened by quenching the fluorescence of coumarin 4-methylumbelliferone (4-MU) through glucosylation. The method enabled to isolate a mutant showing an improved ability for the glucosylation of coumarin 4-methylumbelliferone as well as genistein, daidzein silybin and catechin compared to the parental enzyme [ 210 ].…”

Section: Enzyme Engineering For Man-made α-Glucans Oligosaccharides and Glucoconjugatesmentioning

confidence: 99%

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

et al. 2021

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Glucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.). Usually secreted by their producer organisms, they are involved in the synthesis of α-glucans from sucrose substrate. They contribute to cell protection while promoting adhesion and colonization of different biotopes. Dextran, an α-1,6 linked linear α-glucan, was the first microbial polysaccharide commercialized for medical applications. Advances in the discovery and characterization of these enzymes have remarkably enriched the available diversity with new catalysts. Research into their molecular mechanisms has highlighted important features governing their peculiarities thus opening up many opportunities for engineering these catalysts to provide new routes for the transformation of sucrose into value-added molecules. This article reviews these different aspects with the ambition to show how they constitute the basis for promising future developments.

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“…Transglycosylation reactions of AS are usually conjugations of single and/or multiple (gluco-oligosaccharides) sugar(s) with a great advantage compared with Leloir GTs. For production of bioactive phenolic glycosides [2,3], AS has been used in the synthesis of a variety of attractive biomaterials including amylose-like polymers, starch, dendritic nanoparticles, and microparticle encapsulation [4][5][6]. Although AS has potential application in glycodiversification, it is rarely reported for transglycosyation of phenolic compounds.…”

Section: Introductionmentioning

confidence: 99%

Sustainable Production of Dihydroxybenzene Glucosides Using Immobilized Amylosucrase from Deinococcus geothermalis

Lee¹,

Kim²,

Parajuli³

et al. 2018

Journal of Microbiology and Biotechnology

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The amylosucrase encoding gene from Deinococcus geothermalis DSM 11300 (DgAS) was codonoptimized and expressed in Escherichia coli. The enzyme was employed for biosynthesis of three different dihydroxybenzene glucosides using sucrose as the source of glucose moiety. The reaction parameters, including temperature, pH, and donor (sucrose) and acceptor substrate concentrations, were optimized to increase the production yield. This study demonstrates the highest ever reported molar yield of hydroquinone glucosides 325.6 mM (88.6 g/l), resorcinol glucosides 130.2 mM (35.4 g/l) and catechol glucosides 284.4 mM (77.4 g/l) when 400 mM hydroquinone, 200 mM resorcinol and 300 mM catechol, respectively, were used as an acceptor substrate. Furthermore, the use of commercially available amyloglucosidase at the end of the transglycosylation reaction minimized the glucooligosaccharides, thereby enhancing the target productivity of mono-glucosides. Moreover, the immobilized DgAS on Amicogen LKZ118 beads led to a 278.4 mM (75.8 g/l), 108.8 mM (29.6 g/l) and 211.2 mM (57.5 g/l) final concentration of mono-glycosylated product of hydroquinone, catechol and resorcinol at 35 cycles, respectively, when the same substrate concentration was used as mentioned above. The percent yield of the total glycosides of hydroquinone and catechol varied from 85% to 90% during 35 cycles of reactions in an immobilized system, however, in case of resorcinol the yield was in between 65% to 70%. The immobilized DgAS enhanced the efficiency of the glycosylation reaction and is therefore considered effective for industrial application.

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Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification

Cited by 19 publications

References 62 publications

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

Sustainable Production of Dihydroxybenzene Glucosides Using Immobilized Amylosucrase from Deinococcus geothermalis

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