Recent biotechnological progress in enzymatic synthesis of glycosides

Thuần, Nguyễn Huy; Sohng, Jae Kyung

doi:10.1007/s10295-013-1332-0

Cited by 46 publications

(46 citation statements)

References 176 publications

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“…The enzymes also referred to collectively as Leloir GTs, transfer glycosyl residues from sugar nucleotide donors to specific positions on acceptor substrates (Liang et al, 2015). Compared with other enzymes (e.g., glycoside hydrolases; glycoside phosphorylases) that are also able to glycosylate small molecules, the GTs often present a unique combination of high chemo/regioselectivity and relatively flexible substrate specificity (R. Chen, 2018;Desmet et al, 2012;Thuan & Sohng, 2013). The plant GTs are, therefore, promising catalysts for glycoside production (for reviews, see: Lim, 2005;Nidetzky, Gutmann, & Zhong, 2018).…”

Section: Introductionmentioning

confidence: 99%

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Lemmerer

Mairhofer²,

Lepak

et al. 2019

Biotech & Bioengineering

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Sugar nucleotide‐dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology‐based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l‐arabinose‐inducible expression of the T7 phage‐derived Gp2 protein from a genome‐integrated site. The glycosyltransferase genes are encoded on conventional pET‐based expression plasmids that allow T7 RNA polymerase‐driven inducible expression by isopropyl‐β‐ d‐galactoside. Laboratory batch and scaled‐up (20 L) fed‐batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12‐fold as a result of production under growth‐decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed‐batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3‐fold) and quality (2.3‐fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth‐arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.

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Section: Introductionmentioning

confidence: 99%

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Lemmerer

Mairhofer²,

Lepak

et al. 2019

Biotech & Bioengineering

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“…Therefore, in decades, many efforts have been made to enzymatic modification of stevioside to improve its edulcorant quality through transglycosylation or hydrolysis of stevioside [10][11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

The effect of microwave irradiation on transglycosylation pathway of stevioside with starches or cyclodextrins catalyzed by a cyclodextrin glucanotransferase

Zhang

et al. 2015

Journal of Molecular Catalysis B: Enzymatic

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“…Nature uses an array of different GTs to create and to constantly and specifically change the composition of its diverse display of surface glycans on the surface of the cell. In the laboratory, GTs have been intensively implemented in the synthesis of complex carbohydrates (McArthur and Chen, 2016) and in the glycosylation of small natural molecules (Bowles et al, 2005; Thuan and Sohng, 2013). However, the use of these novel methods to engineer glycans in living cells has been limited.…”

Section: Introductionmentioning

confidence: 99%

Labeling glycans on living cells by a chemoenzymatic glycoengineering approach

Almaraz

2017

Biology Open

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Structural glycobiology has traditionally been a challenging field due to a limited set of tools available to investigate the diverse and complex glycan molecules. However, we cannot ignore that glycans play critical roles in health as well as in disease, and are present in more than 50% of all proteins and on over 80% of all surface proteins. Chemoenzymatic glycoengineering (CGE) methods are a powerful set of tools to synthesize complex glycans, but the full potential of these methods have not been explored in cell biology yet. Herein, we report the labeling of live Chinese hamster ovary (CHO) cells by employing three highly specific glycosyltransferases: a sialyltransferase, a galactosyltransferase, and an N-acetyl-glucosaminyl transferase. We verified our results by bio-orthogonal blots and further rationalized them by computational modeling. We expect CGE applications in cell biology to rise and their implementation will assist in structural-functional discoveries in glycobiology. This research will contribute to this effort.

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Recent biotechnological progress in enzymatic synthesis of glycosides

Cited by 46 publications

References 176 publications

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases

The effect of microwave irradiation on transglycosylation pathway of stevioside with starches or cyclodextrins catalyzed by a cyclodextrin glucanotransferase

Labeling glycans on living cells by a chemoenzymatic glycoengineering approach

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