Successes in engineering glucansucrases

to enhance glycodiversification
a
a
a
David Daude
, Isabelle Andre
, Pierre Monsan and
b,c
Magali Remaud-Sime
on*

DOI: 10.1039/9781849739986-00624

Carbohydrates are biomolecules that have an essential role in every form of life. The
reservoir of naturally occurring glyco-structures is incredibly large and involves a tremendous number of carbohydrate-active enzymes (more than 280,000 released modules
in the Carbohydrate Active enZymes database) for their synthesis and degradation.
Nevertheless, natural enzymes do not necessarily present all the requested properties in
terms of eciency, specificity or stability when considering their usage for carbohydrate
or glyco-derivative manufacturing. In addition, if existing, the identification of an enzyme
perfectly adapted to a specific function from the natural diversity may be critical due to the
lack of available biochemical data and may necessitate intensive screening eorts. To
circumvent such limitations and provide optimized solutions, protein engineering has
been considered. Leloir-type glycosyltransferases, for example, are mainly involved in the
biosynthesis of glycoconjugates in Nature and they have been widely studied and engineered for this purpose. However, these enzymes are often found as membrane-bound
proteins, what renders dicult their isolation and purification. In addition, their need of
low-abundant activated sugars as glycosyl donors also impairs their usage. Alternatively,
enzymes that use more abundant glycosyl donor directly issued from agro-ressources
have been considered to access to new glyco-derivatives. This has promoted the use of
glucansucrases (GS) that catalyze transglycosylation reactions from sucrose substrate.
These enzymes are of particular interest for synthetic purpose and have found industrial
interest for pharmaceutical and fine chemical applications. To diversify their applications,
various approaches of engineering have been exploited to improve expression level,
stability, or change substrate or product specificity of these enzymes. In particular, the
range of molecules recognized and the osidic linkages formed by GS is broad but yet
limited. Therefore, protein engineering methods have been applied to further increase the
diversity of glycosylation reactions catalyzed by these enzymes. Sequence analysis and
mutagenesis experiments have enabled the identification of key amino acid residues of
glucansucrases either involved in catalysis or substrate specificity. Moreover, the determination of three-dimensional structures of glucansucrases from both families 13 and
70 of Glycoside-Hydrolases (GH) have provided powerful information for understanding
the sequence-structure-function relationships and guiding structure-based rational and
semi-rational engineering of these proteins. To assist these eorts, high-throughput
screening and biomolecular methods have been developed for the directed evolution of
these enzymes. Here are reported some of the successes in the bioengineering of glucansucrases from precursor work to latest results, as well as the methods developed for
screening and developing ecient variant libraries. The major progresses and breakthroughs in the field will be highlighted and further prospects will be considered and
discussed.

Universite de Toulouse; INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077

Toulouse, France
b
CNRS, UMR5504, F-31400 Toulouse, France
c
INRA, UMR792 Ingenierie des Syste`mes Biologiques et des Procedes, F-31400
Toulouse, France. E-mail: magali.remaud@insa-toulouse.fr
624 | Carbohydr. Chem., 2014, 40, 624645

c

The Royal Society of Chemistry 2014

Introduction

Glucansucrases (GS) are a-transglucosylases involved in a-glucan biosynthesis. These enzymes have been classified as glycosyltransferases by
the Enzyme Commission (EC 2.4.1.4, EC 2.4.1.5, and EC 2.4.1.140) and
placed in the category of Glycoside-Hydrolases (GH) by the CAZy classification according to their sequence and structure similarities.1 Most
glucansucrases (dextransucrases, alternansucrases, reuteransucrases and
mutansucrases) belong to the GH70 family and synthesize a-glucans
harbouring various osidic linkages (Fig. 1B) with the exception of
amylosucrases, responsible for the synthesis of an amylose-like polymer,
which are part of the GH13 family. Unlike Leloir-glycosyltransferases,
glucansucrases do not require activated nucleotide-sugars as donor
substrates as they use sucrose, which contains an osidic linkage with an
energetic level similar to the one of nucleotide-sugars. From sole sucrose,
GS naturally catalyze the synthesis of a-glucans, as well as the glucosylation of hydroxylated acceptors such as gluco-oligosaccharides, sucrose
or fructose (Fig. 1B). They follow an a-retaining mechanism involving
first the formation of a b-D-glucosyl-enzyme covalent intermediate with a
concomitant release of fructose (Fig. 1A). In a second step, this intermediate is attacked by the hydroxyl group of an acceptor to release the
glucosyl residue. Glucan synthesis occurs via successive transfers of the
glucosyl units onto glucooligosaccharides. Depending on the enzyme
specificity, the produced polymers dier in terms of size as well as
number, type and arrangement of osidic linkages.
The scope of reactions catalyzed by glucansucrases can also be extended by introducing exogenous hydroxylated molecules into the reaction media which, if recognized by the enzyme, can play the role of
acceptor in glucosylation reactions. A variety of oligosaccharides or glucoconjugates can thus be synthesized depending on the nature of the
considered acceptor and the enzyme specificity. A wide range of acceptors has been reported for GS belonging to GH70 family and the main
ones are shown in Fig. 2. Molecules from various types can be recognized
by glucansucrases, ranging from monosaccharides structurally similar to
the natural acceptor to unconventional substrates such as amino acid
derivatives or bulky flavonoids. Amylosucrases from GH13 family are also
known to glucosylate diverse acceptors, albeit they have been less investigated for this purpose (Fig. 3). Though several acceptor substrates
have been identified, only a small number of donors have been reported
to date. Fluoro-glucosides, p-nitrophenyl-a-D-glucopyranoside and maltooligosaccharides have been shown to act as glucosyl donors for amylosucrases.24 Interestingly, some sucrose derivatives harbouring a
modified glucosyl moiety have been described as possible substrates for
transglycosylation reactions. The 2-ketosucrose for example has been
identified as an alternative donor substrate of glucansucrases for synthesizing carbonyl-group-containing dextran.5 The a-D-galactopyranosyl1,2-b-D-fructofuranoside and allo-sucrose have also been mentioned as
potential donors for amylosucrases, even if further characterization may
be required.6,7 Nevertheless, most sucrose derivatives and analogs
Carbohydr. Chem., 2014, 40, 624645 | 625

626 | Carbohydr. Chem., 2014, 40, 624645

OR

-O

glucosylation

O
H

O
O

11

13

Sucrose

(12)

Hydrolysis

O
-O

15

Leucrose

Turanose

O
H

Glucose

12

16

Fructose

16

14

16

14

16

16

13

16

13

13

16

Polymerization

14

13

16

16

12
16

14

13

16

16

14

HO

OR

-O
O

Non-saccharidic acceptor

-1,2 branched glucan

Reuteran

Mutan

Dextran

Alternan

Amylose

R = Fructose
2
R = H : Hydrolysis
2
R = Carbohydrate : Transglucosylation
2
R = Fructose : Sucrose isomerization

deglucosylation

Monosaccharide

16

16

13

16

13

14

Transition state

14

GH70 products

14

GH13 product

Covalent -D-glucosyl enzyme

Trehalulose

R2OH R1OH

Sucrose isomerization

Transition state

Fig. 1 Reactions catalyzed by glucansucrases. (A) General mechanism; (B) Overview of the products generated by glucansucrases.

Glucoconjugates

Acceptor reactions

Oligosaccharides

Acidic/basic residue (Glu)

HO

Nucleophilic residue (Asp)

OH

OH
O

HO
HO

H 3C
HO

OH

HO

NHAc

N-Acetyl-D-glucosamine

HO
HO

OH

OH

-D-Tagatose OH

1,5-Anhydro-D-fructose

[41]

[42]

OH

HO

OH

OH

OH

Methyl -D-Glucopyranoside

OCH 3
OH

Methyl -D-Glucopyranoside

L-Glucose

[43]

HO
HO

OH

OH
OCH 3

[43]

OH
O

HO
HO

OH
O

HO
HO

OH

L-Rhamnose

[41]

OH
O

OH

[44, 45, 46]

[44, 45]

HO OH

OH
HO OH

HO
OH
OCH 3

HO
OCH 3

HO

OCH 3

OH

Methyl -D-Galactopyranoside

Methyl -D-Mannopyranoside

Methyl -D-Galactopyranoside

[44]

[44]

[44]

OH
OH

OH

HO

OH
O

HO
HO

OH
O

HO

HO

OH
OCH 3

OH

Methyl -D-Mannopyranoside

OH

Methyl -D-Allopyranoside

[44]

OCH 3

OH

OCH 3

Methyl -D-Allopyranoside

[44]

[44]

OH
OCH 3
H 3C
HO

HO
HO

OH
O(CH 2 ) n CH 3

HO

OH

Methyl -L-Rhamnopyranoside

Alkyl -D-Glucopyranoside

[41]

n=0, 3, 7...

[47]

OH
OH
HO
HO

HO
HO

OH O

OH
OH
O

O
HO
OH

Maltose

HO
HO

OH

[233, 236-238]

OH

OH

Isomaltose
[45, 48]

OH
HO
HO

HO OH
O

OH
O

OH

HO
OH

Gentiobiose
[49]

HO

OH
OH

HO

HO

OH

OH

Lactose
[50]

Fig. 2 Representation of some exogenous acceptors recognized by glucansucrases from

GH70 family to catalyze transglucosylation reactions.4167

Carbohydr. Chem., 2014, 40, 624645 | 627

OH

HO

HO

HO
HO

HO

OHOH

O
OH

OH

OH

O
O

OH

OH

HO
OH

Maltulose

Lactulose

OH
[48]

[51]

OH
OH
HO
HO

OH
O

OH HO
O

O
HO

OH

HO
HO

OH
O

OH

OH

OH

OH

Nigerose

Cellobiose
[52]

[45]

HO OH
O
HO
OH

HO
HO

OH
O

Raffinose

HO

OH
O

OR

HO
OH

HO

6-O-Tosyl-glucose derivatives
OH

OH

R = H, Me(), allyl ()

[53]

[54]

OH
O

HO
HO
OH

OH

OH

OH

OH

OH
HO

OH

HO
OH

HO

OH

OH

OH

OH

D-Mannitol

D-Sorbitol
[48, 55]

OH

D-Maltitol

[48]

[48, 55]

OH

OH
HO
HO

OH

OH

O
OH

HO
HO

OH

O
OH

OH

OH

OH
OH

OH

OH

OH

OH

-D-Glucopyranosyl-(1,6)- D-sorbitol
[48]

[48]

Fig. 2 (Continued)

628 | Carbohydr. Chem., 2014, 40, 624645

OH

-D-Glucopyranosyl-(1,6)- D-mannitol

OH

OH
OH

HO
HO

HO O

HO

OH

OH
OH

HO

OH

OH

OH

Trehalulose

-D-arabino-Hexos-3-ulopyranosyl-(1,6)- D-fructose

OH
[48]

[48]

OH

OH
HO
HO

O
OH

HO
HO

OH

OH

OH

O
OH

OK
OH

OH

-D-arabino-hexos-3-ulopyranosyl-(1,6)-D-mannitol

[55]

HO

OH

OH

-D-Glucopyranosyl-(1,6)-D-arabinonic acid

[48]

HO

CH 2 OH

HO

HO
HO

OH

O
OH

O
O

OH

OH

OH

-D-Fructofuranosyl- -D-fructofuranosyl-(1,2':2,3')-dianhydride

Salicin
[246]

[48]

OH

HO
HO
OH
HN

O
OH

HO
OH

O
O
HO

OH
OH
O

O
HO

Acarbose

OH
OH

OH
[56]

HO
HO

OH
OH

O
HO
OH
O

n-decyl [-D-Glucopyranosyl-(1,4)-] -D-glucopyranoside

[48]

OH

OH

OH

OCH 3
BocHN
O

OCH 3
BocHN

OH

N-tert-butoxycarbonyl- N-tert-butoxycarbonylN-tert-ButoxycarbonylL-serine methyl ester

L-threonine methyl ester D-serine methyl ester
[57]

OH

BocHN

[57]

[57]

HO
HO

D-glucal
[48, 55]

Fig. 2 (Continued)

Carbohydr. Chem., 2014, 40, 624645 | 629

Cl

(CH 2 )

OH

H 3C

(CH 2 )

n=2, 4, 6

OH
n

n=0, 1, 2, 3, 4...

Primary alcohols
(methanol, ethanol, propanol, butanol...)

Chloro derivatives
(2-chloroethanol, 4-chlorobutanol, 6-chlorohexanol)
[57]

[57, 58]

OH
OH

OH

HO

HO

OH

OH

OH

OH
OH

OH

Myricetin

Quercetin
[59, 60]

[60]

OH

OH

OH

HO

HO

OH

OH

OH

OH

OH

Luteolin

Ampelopsin

[60]

[61]

OH
OH

OH

HO

OH
OH

OH
O

O
HO

OH

O
OH

OH

HO

OH
O

Epigallocatechin gallate

Astragalin
[62]

OH

[63]

HO

HO

OH

COOH
HO

NH2

OH

HO

Phenol

Salicyl alcohol

[64]

[64]

OH

L-DOPA

OH

Catechol

[65]

[66]

HO

OH

OH

H
O

HO

OH

OH

OCH 3

3-Methoxycatechol
[66]

H 3C

CH 3

4-Methylcatechol

3-Methylcatechol
[66]

OH

[66]

Fig. 2 (Continued)

630 | Carbohydr. Chem., 2014, 40, 624645

HO

L-ascorbic acid
[67]

OH

assessed for transglycosylation purpose were not recognized as substrate,

some of them even acting as inhibitor.811 The use of sucrose derivatives
by natural or engineered enzymes would be challenging for enhancing
glycodiversification while using non-activated carbohydrates.1227
Glucansucrases display both a tremendous promiscuity toward acceptor substrates and an increased specificity for sucrose that may be
related to their three-dimensional organization. Indeed crystal structures
of glucansucrases from both GH13 and GH70 families have been obtained in either apo or holo forms and they constitute an incredible
source of information for biochemical and biophysical investigations of
GS which have been very well reviewed by Leemhuis et al.2836 These
enzymes, although displaying distinct three-dimensional organizations
(U-shape like structure for GH70 and TIM-barrel for GH13), as illustrated
in Fig. 4A, share similar active-site topology (Fig. 4B).3739 Two subsites,
namely  1 and 1 according to GH nomenclature,40 are responsible for
sucrose and acceptor recognition. Subsite  1 is involved in the strong
anity for sucrose via a dense polar network of highly specific interactions for its glucosyl moiety. Subsite 1 is both involved in acceptor
and sucrose fructosyl recognition and therefore, it shows a tremendous
plasticity that enables glucosylation of various hydroxylated molecules.
These subsites are responsible for both reactivity and substrate
accommodations and are thus highly determinant for the biocatalytic
properties of glucansucrases.
Although glucansucrases naturally dispose of very interesting properties in terms of substrate utilization, catalytic promiscuity or product
diversity, protein engineering has been considered to further optimize
these biocatalysts and further extend their potential for glycodiversification, as well as their compatibility to industrial processes. About
20 years of scientific investigations in the field have enabled to generate
performing enzymes which display unprecedented properties. An overview of these achievements is proposed here, from pioneering work to
very recent successes in glucansucrase engineering. The range of tailored
screening methodologies is described and future challenges and prospects will be highlighted and discussed.

2 Random approach for glucansucrase overproduction

or engineering
2.1 Generation of mutants of native producing strains
By the end of the 90s, some precursor studies started to investigate the
role of genetic modifications of native glucansucrase producing strains.
Chemical mutagenesis using ethyl methane sulfonate has been extensively used for generating constitutive variants of glucansucrases. Various
strains of Leucosnostoc mesenteroides B-512FM, B-1299, B-1142, B-1299,
B-742 and Lm 28 were engineered in this way. Variants B-1355C and
B-1142C were able to produce glucans highly resistant to Penicillium
dextranase hydrolysis.74,75 Mutant B-512FMC produced the same kind of
dextran as the parental B-512FM but showed higher thermal stability.74,75
Dextran produced by variants B-1299C, B-1299CA and B-1299CB diered
Carbohydr. Chem., 2014, 40, 624645 | 631

OH
OH

HO
H 3C

HO
HO

OH

OH

HO
OH
O

HO

OH

HO

NHAc

N-Acetyl-D-glucosamine

OH

[41]

[41]

[68]

OH

HO
O

HO

HO
HO

HO

OH
OH

Maltose

L-Rhamnose

OH

O
HO

OH

OH
OH

Arbutin

Salicin
[69]

[70, 71]

OH

OH
OH

HO
HO

HO
HO

O
OH

OH
HO

Piceid

Aesculin

OH

[71]

[72]

HO

HO

O
OH

HO

Hydroquinone

Caffeic acid

[72]

HO

OH

OCH 3

Vanillin

OH

[71]

Zingerose

[71]

[71]

OH

HO

OH

OH

OH
O

HO

O
HO

OH

OH

OH

(+) Catechin

D-Arabinose

[73]

OH

OH

OH

OH
OH
O

HO
HO

HO
OH

OH

OH

[74]

[74]

HO

OH

OH

OH

L-Arabinose
[74]

OH
OH OH

OH

D-Allose

OH
O

OH

HO

[74]

[74]

HO O

HO

OH

D-Xylose

[74]

O
OH

OH

D-Altrose

OH

OH

HO
HO

OH

D-Mannose

OH

[74]

OH
OH
O

HO

OH

D-Galactose

HO

D-Fucose

[74]

OH

OH

L-Fucose
[74]

L-Galactose
[74]

Fig. 3 Representation of some exogenous acceptors recognized by glucansucrases from

GH13 family to catalyze transglucosylation reactions.6873

632 | Carbohydr. Chem., 2014, 40, 624645

HO

HO

OH

O
OH

OH
OH OH

HO

L-Mannose

OH

OH
OH

OH

L-Xylose

L-Altrose
[74]

OH

OH
O

HO

OH

OH
OH

[74]

HO

OH
O

L-Allose

[74]

[74]

OH

OH

OH

OH

OH

HO

OH
HO

OH

OH
HO

OH

OH

OH

D-Sorbitol

OH

OH

D-Arabitol

D-Mannitol

[74]

[74]

[74]

OH
O

HO
HO

OH
OH

O
OH

HO

OH
OH

HO

HO OH
HO

OH
OH

HO
OH

OH

D-Xylitol
[74]

OH

OH

D-Maltitol
[74]

Myo-inositol
[74]

Fig. 3 (Continued)

from the glucan produced by the native strain in terms of solubility or

susceptibility to endo-dextranase hydrolysis.76 Variants B-742CA and
B-742CB synthesized dextran with a-1,4 branched linkages and a high
percentage of a-1,3 bonds, respectively.77 Mutants Lm M281 and Lm
M286 derived from L. mesenteroides strain Lm 28 were shown to produce
more active glucosyltransferases or resistant glucan, respectively.78 Synchrotron radiations in the 701000 eV region were also considered for
further engineering the Leuconostoc mesenteroides B-12FMC variant.79
From this work, a hyperproducing mutant constitutive for dextransucrase, namely B-512FMCM, was shown to produce 13-fold increase
in activity and 1000-fold increase in glucansucrase protein compared to
the parental strain. UV radiations were also used to engineer the dextransucrase producing strain Leuconostoc mesenteroides KIBGE IB-22. One
mutant over the 42 generated showed a 6.75 fold increase in activity
compared to the wild-type enzyme.80
Altogether, these results underline that genetic engineering of native
strains may be useful for both increasing the production of glucans and
modulating their properties.
2.2 Random engineering of recombinant enzymes
Because native strains may be sometimes dicult to handle, the use of
recombinant enzymes produced into well-known organisms such as
Escherichia coli has been investigated. Genes coding for glucansucrases
have been isolated, heterologously expressed and further considered for
bioengineering experiments. Ultrasoft X-ray irradiations have been
applied to an Escherichia coli transformant to generate dextransucrase
Carbohydr. Chem., 2014, 40, 624645 | 633

Amylosucrase from N. polysaccharea (GH13)

GTF180-N from L. reuteri (GH70)

1 N

746

Domain V

Domain N

1751

90

Domain B
184

793

1639

Domain IV
260

Domain B
927

1605

990

1591

Domain B
395

460

Domain A

550

Domain C

Domain A
1238

636

1377

Domain C

B
GH13 amylosucrase from N. polysaccharea (PDB 1G5A)
R446

GH70 glucansucrase GTF180-N from L. reuteri (PDB 3KLK)

Subsite +1

D394

Subsite +1
D1136

R509
D393

Q1140

W1065

H1135
D507

N1411
H392
E328
F250

D144
R284

R1023

D1458

E1063

D1025

D286
Y1465

Y147
Subsite -1

H187

Subsite -1

D1504
Q1509

Fig. 4 Structural comparisons of glucansucrases from GH13 and GH70 families. (A)
Overall three-dimensional structure of amylosucrase from N. polysaccharea (pdb:1G5A)
and glucansucrase GTF180 from L. reuteri (pdb:3KLK). (B) Representation of subsites  1
and 1 constituting the bottom of active site pocket, docked with sucrose (extracted
from pdb:1JGI and pdb:3HZ3, respectively). Subsites of glucansucrases from both GH13
and GH70 families share a similar spatial organization and involve comparable hydrogen
bonding networks mainly due to subsite  1. Catalytic residues are highlighted in bold and
water molecules are repredented as spheres.

variants of DSRB742 with increased constitutive activity for the synthesis

of highly a-1,3 branched dextran.81 The same procedure was applied to
generate the novel dextransucrase gene DSRN and was further combined
with site-directed mutagenesis to construct four mutants (DSRN1 to
DSRN4), one of them DSRN3 (K395T) showing the highest transglucosylation eciency with diverse acceptors (maltose, salicin, gentiobiose).82
634 | Carbohydr. Chem., 2014, 40, 624645

Along with these reports, combinatorial engineering have been attempted to increase the specific activity of glucansucrases. The performance of amylosucrase from Neisseria polysaccharea (NpAS) was
enhanced by random mutagenesis, gene shuing and selective screening. Variants with up to a five fold increase in activity toward sucrose were
isolated (R20C/F598S and V389L/N503I).83 Variants with increased polymerization eciency (E227G), thermostability (P157A/D231Y, P234L/
G554S and N387D) or activity (N76D, E62K/D506N, N387D, Q613H) were
further isolated.84 Random engineering strategies may thus be useful for
enhancing the performances of recombinant enzymes what is of prime
interest for biotechnological purposes. Moreover, industrial processes
often require high reaction temperatures and the enhancement of enzyme thermostability is still challenging. Directed evolution of NpAS by
error-prone PCR has been performed and led to the isolation of two
double mutants (R20C/A451T and A170V/Q353L) and a single mutant
(P351S) with 3.5 up to 10 fold increased half-lives at 50 1C as compared to
the parental wild-type enzyme. The increased stability was suspected to
be due to the introduction of additional hydrogen-bonding interactions
and salt-bridge rearrangements that are assumed to strengthen the
overall structure.85

Structure-based engineering of glucansucrases

3.1 Scaold diversification

Glucansucrases are usually large proteins (W120kDa except amylosucrase
(70kDa)) which can hinder their ecient production. To circumvent this
limitation, the construction of truncated variants was considered to
generate more stable and more soluble proteins. A truncated variant of
alternansucrase from Leuconostoc mesenteroides NRRL B-1355, with
modified repeating units of the C-terminal domain was constructed and
showed the same specificity as the native enzyme while being highly
active.86,87 Mutants rationally shortened of their signal peptide have also
been constructed and resulted in ecient, active and stable recombinant
glucansucrases.8890 Truncated forms of DSRE563, a dextransucrase obtained from the constitutive mutant CB4-BF563 derived from L. mesenteroides B-1299, were constructed. These trimmed enzymes DSRE563-1
and DSRE563-2 were shown to synthesize a less-soluble dextran.91
Glucansucrases such as DSR-E produced by L. mesenteroides NRRL
B-1299 are known to display original structures harbouring two active
sites. In this case, Catalytic Domain 1 (CD1) synthesizes a-1,6 linkages,
while Catalytic Domain 2 (CD2) is responsible for the rare and unusual
a-1,2 specificity. Truncated forms of this protein have been constructed.
CD2 domain was deleted to produce the engineered DSR-E-D(CD2)
enzyme responsible for the synthesis of dextran containing mainly a-1,6
linkages, as well as a-1,3 and a-1,4 linkages but no a-1,2 bond.
Conversely, the truncated form of the first catalytic domain GBD-CD2
(DSR-E-D(VZ-CD1)) after construction, turned out to be unable to catalyze
polymerization reactions and only hydrolytic reactions were observed.
Nevertheless, using sucrose as donor and isomalto-oligosaccharides as
Carbohydr. Chem., 2014, 40, 624645 | 635

acceptors, the GBD-CD2 enzyme was able to catalyze transglucosylation

reactions and interestingly, it synthesized solely a-1,2 linkages.92 The
mode of branching of this enzyme was investigated through the analysis
of a 1.5 kDa grafted dextran and revealed a stochastic branching
process.93
In order to provide better insights on the sequence-structure-function
relationships of glucansucrases, attempts have been made to diversify
their three-dimensional organization by varying enzyme scaolds. In vitro
constructions of chimeric glucansucrases have been attempted. Selected
sites of glucansucrases DSRS and DSRT5 from Leuconostoc mesenteroides
NRRL 512-F have been exchanged and six chimeric variants were constructed. Upon analysis, their products were found to dier from the
glucans synthesized by their parental enzyme in terms of solubility and
linkage specificity.94 Fusion proteins DXSR harbouring dextransucrase
and dextranase activities were generated and successfully expressed in
E. coli and used for the production of linear isomalto-oligosaccharides
(IMO) further increased by the introduction of metal ions to reach a
30-fold increase in the production of IMO as compared to a mixture of the
two enzymes.95 Another fusion protein namely DSXR has also been
constructed and the expression level was optimized using response surface methodology to overcome the low productivity of DXSR but conserving similar properties.96 Fusion protein involving glucansucrases
were also considered for transgenic investigations. Dextransucrase DSR-S
from Leuconostoc mesenteroides fused to the chloroplastic ferredoxin
signal peptide was used to transform two potato genotypes (cv. Kardal
and the amylose-free mutant (amf)). Dextrans were detected in potatoes
tuber juices from transformants of both species but with a two fold increased concentration for Kardal. No dextran could be detected inside the
starch granule, however the morphology of this latter was altered probably due to an accumulation of dextran in the tuber juices.97 A truncated
mutansucrase GtfICAT without starch-binding domain derived from GtfI
was expressed in Kardal. The production of mutan adhering to starch
granules was detected but not incorporated in the starch granules.98 A
fusion protein comprising GtfICAT and a starch-binding domain (SBD) at
either N- or C- terminal end was introduced in two genetically dierent
potato backgrounds. The fusion protein was detected in starch granules.
Starches from the plant expressing GtfICAT contained less mutan than
GtfI expressing plant. However, the granule morphology was altered in
both genetic backgrounds. These results underline the fact that
expression of engineered glucansucrases can be used to interfere with
starch biosynthetic pathway in plants.99
3.2 Semi-rational and rational engineering
Glucansucrases are mainly involved in the synthesis of a wide variety of
gluco-oligosaccharides and high molecular weight glucopolymers. Determination of the three-dimensional structures of glucansucrases have
enabled to investigate the role of the dierent subsites and glucan
binding domains of the enzymes. Intensive work has shown that properties of the glucan synthesized such as specificity of the osidic linkages
636 | Carbohydr. Chem., 2014, 40, 624645

or chain length are enzyme-dependent and may be modulated through

protein engineering.
Mutagenesis experiments of Q937 and D569 positions of glucansucrase
GTF-I from Streptococcus downei showed that single amino acid substitutions can impact the glucan linkage.100,101 Another enzyme, dextransucrase DSRS from Leuconostoc mesenteroides NRRL B-512F, has been
submitted to site-directed mutagenesis. Lysine residues were introduced
at the N-terminal end. Two single mutants, T350K and S455K, and the
corresponding double-mutant T350K/S455K were constructed. Their
products showed an enhanced amount of 1,6-linked Glcp going from 70
to 85% for the single mutants and the unusual presence of 2,6-linked
Glcp for the double-mutant.102 Recent semi-combinatorial engineering of
this enzyme has also underscored the impact of amino acid mutations on
glucan structures and properties. Eight residues from the catalytic domain were targeted from sequence analysis and engineered using Incorporating Synthetic Oligos via Gene Reassembling (ISOR) method.103
Products obtained using the variants of a truncated DSRS (DSRS vardel
D4N) harbouring from one to four amino acid substitutions (F353T,
S512C, F353W, H463R/T464D/S512C, H463R/T464V/S512C, D460A/
H463S/T464L, D460M/H463Y/T464M/S512C) were analyzed and revealed
polymers diering in their a-1,3 linkage contents and their gel-like
properties in solution. This work provided a useful toolbox of glucansucrases producing increasing amounts of a-1,3 linkages.104
Reuteransucrase from Lactobacillus reuteri 121 GTF-A was also successfully engineered. The role of N1134 located right next to the catalytic
residue D1133 was investigated and it was shown to be involved in both
product specificity and hydrolysis/transglucosylation ratio. Single mutants at this position aected the total specific activity going from
4575% loss for N1134Q, N1134G or N1134H up to two fold increase for
N1134A, N1134D and N1134S.105 Another study on this enzyme converted
its reuteransucrase activity into a dextransucrase activity by increasing
the amount of a-1,6 linkage from B35% up to B85% and decreasing the
amount of a-1,4 linkages from B45% down to B5% for the quintuple
mutant P1026V/I1029V/N1134S/N1135E/S1136V. These results underline
the role of amino acid changes in enzyme mechanism and product
nature.106
Bioengineering of glucansucrase GTF-180, an a-1,3/a-1,6 linkage synthesizing enzyme, from Lactobacillus reuteri strain 180 was undertaken
leading to the creation of a triple mutant V1027P/S1137N/A1139S able to
synthesize a-1,4 linkages. Twelve other variants were also identified as
producing modified exopolysaccharides, some of them generating a-1,4
linkages. The products synthesized by these variants were analyzed and
showed discrepancies in their structural and physical properties such as
solubility, molecular weight or structure.107,108
Specificity of glucansucrase GTF-R from Streptococcus oralis was also
modulated through random mutagenesis of a conserved motif surrounding the transition state stabilizer.109 A triple mutant R624G/V630I/
D717A was identified as producing a mutan type polymer harbouring
mainly a-1,3 linkages by opposition to the wild-type produced dextran
Carbohydr. Chem., 2014, 40, 624645 | 637

type polymer which is mainly composed of a-1,6 linkages. Mutagenesis at

position S268 was also applied what led to variants with modified
transglucosylation properties. Variants mainly synthesizing short-chain
oligosaccharides, among which mutants S268D and S268R, lost their
capacity to synthesize polymers probably by facilitating the release of
acceptor reaction products as well as the attack of GTFR-Glc intermediate
by water and acceptor substrates.109
Amino acid residues located near the active site of DSRBCB4 dextransucrase from Leuconostoc mesenteroides B-1299CB4 were targeted by
site-directed mutagenesis. The triple mutant V532P/E643N/V644S was
constructed and showed to add a-1,3 and a-1,4 linkages onto the a-1,6
linked glucan mainly synthesized by the wild-type enzyme. The V535I/
S642N mutations were subsequently introduced by directed mutagenesis.
The resulting variant was shown to synthesize an increased amount of
a-1,4 linkages (up to 11%) compared to the triple-mutant.110
The molecular basis of glucan production of amylosucrase from Neisseria polysaccharea was also investigated through the engineering of
subsites 1, 2, 3 residues. An outstanding variant R446A was isolated which synthesizes twice as much insoluble glucan as the parental
enzyme while generating lower amounts of by-products.38
The use of glucansucrases might be impaired by uncontrolled levels of
sucrose hydrolysis which is a minor reaction occurring naturally. The
transglucosylation/hydrolysis ratio has thus to be considered when optimizing performances of GS in order to limit their side reactions. Hybrid
reuteransucrases have been constructed in this way. Some variants exhibiting strongly increased transglucosylation activities were obtained by
targeting specific regions of the catalytic domains. The conversion of
sucrose into oligosaccharide and polysaccharide products was increased.
Two variants namely GTFO-A-dN and GTFO-dN-RS, derived from the
reuteransucrase GTFO from Lactobacillus reuteri ATCC55730, displayed
reduced hydrolysis activities as compared to their parental enzyme but
maintained the a-1,4 linkage specificity.111 The transglucosylation/hydrolysis ratio appears thus to be controllable through rational protein
engineering.
Though mainly used for glucan synthesis, glucansucrases can also be
exploited for synthesizing glucoconjugates or short oligosaccharides,
polymerization being then undesired for this purpose. The polymerization capacity of GS may be modulated to favour the production of
short glucosylated products. The gene encoding the amylosucrase from
Neisseria polysaccharea was submitted to high-rate segmental random
mutagenesis. A segment coding for amino acids involved in substrates
recognition was targeted. Two residues, D394 and G396 were identified as
playing a major role in the control of generated chain length. Indeed, by
substituting these residues with bulky amino acids, the synthesis of short
oligosaccharides (up to three units) was shown to be favoured. Steric
hindrance introduced at these sites was thought to interfere with the
elongation of amylose chains. The variants selected were specific of the
synthesis of mono and di-glucosylated products and could be considered
for the limited glucosylation of acceptors.112
638 | Carbohydr. Chem., 2014, 40, 624645

As described earlier in this manuscript, GS are known to glucosylate

more or less eciently a wide range of hydroxylated molecules. The efficiency of this non-natural reaction will strongly depend on how well the
exogenous acceptor is able to compete with the natural products resulting from sucrose utilization and present in the reaction media. The
development of biocatalysts with enhanced or even new glucosylation
capabilities is thus challenging and has only scarcely been considered.
Mutagenesis experiments applied to the amylosucrase from Neisseria
polysaccharea have been recently used to improve the acceptor glucosylation rate using sucrose as donor. In this report, seven residues were
targeted for saturation mutagenesis and 133 mono-variants were
constructed. The eciency of the glucosylation of 2-acetamido-2-deoxy-aD-glucopyranoside was remarkably enhanced by some single mutants to
reach conversion degrees over 90% which were accompanied by up to
130-fold enhanced catalytic eciency. This library was also assayed
toward another molecule, the methyl-a-L-rhamnopyranoside, non-recognized by the wild-type enzyme. Fifteen mutants harbouring mutations at
either positions 228 or 290 displayed a remarkable novel specificity
toward the exogenous acceptor and they were able to glucosylate it with
remarkable conversion degrees going up to 44% after protein purification.113,114 The pairwise recombination of these mutations was further
applied and led to the isolation of several double mutants displaying a
spectacular 400-fold improvement of their catalytic eciency toward
2-acetamido-2-deoxy-a-D-glucopyranoside.115 A structure-based engineering of this amylosucrase using stability change predictions was recently
reported.22 The reshaping of subsite  1 was investigated leading to the
evaluation of 57 single mutants. Some variants were found more stable
than the wild-type enzyme or able to synthesize a series of oligosaccharides
with original distribution profile. Protein engineering appears to be
noteworthy for developing novel glucansucrases with unprecedented
properties that need to be further investigated in this purpose.

4 Screening methods applied to detect novel or

improved glucansucrases
Enzyme engineering strategies often require generating an important
number of variants and positive selection pressure usually have to be
applied for isolating original mutants from large libraries. Highthroughput screening methods have been developed to assay glucansucrase libraries. Automated protocols have been proposed for the isolation
of amylosucrase variants with improved biochemical properties such as
thermostability or organic solvent tolerance or activity. These methods
enabled already the identification of mutants generated by random
mutagenesis approach using a human mutagenic DNA polymerase displaying up to 25-fold increased activity at 50 1C as compared to the parental NpAS.116,117 Variants with increased activity (up to 5-fold) were
identified using automated reducing sugar assay.83 Methods for the
isolation of eective transformants displaying desired properties have
also been developed. A pH sensitive high-throughput screening has been
Carbohydr. Chem., 2014, 40, 624645 | 639

used for the selection of sucrose-utilizing transglycosylases. E. coli competent cells unable to use sucrose, transformed by a plasmid containing
an engineered gene coding for an amylosucrase activity, were grown on
solid medium containing sucrose and bromothymol blue (BTB) as pH
colour indicator. Cells expressing an active amylosucrase variant were
able to use sucrose and synthesize an amylose-like polymer. The fructose
released during the polymerization reaction was metabolized by E. coli
cells through glycolysis pathway to synthesize acidic products, the local
acidification being detected by BTB color change from blue to yellow.118
Another method has been developed for isolating E. coli cells displaying
intracellular dextransucrase activities that can be identified through a
polymer-forming based strategy. E. coli transformants are grown on solid
medium supplemented by 2% of sucrose. Clones displaying dextransucrase activity synthesize an extracellular glucan and can thus be
detected.119 Recently, a powerful medium-throughput screening of glucansucrase specificity has been developed. Product specificities of more
than 4,000 glucansucrase variants generated by combinatorial engineering were screened through a quantitive and highly sensitive NMR
based-approach with a rate of 480 variants per day. Altogether, 303
variants were successfully identified for their altered specificity underlining the potential of this method in glycomics for screening natural
glucan biodiversity.103 Surface Plasmon Resonance spectroscopy has
also been used for the detection of transglycosylase-catalyzed polymer
synthesis and the determination of enzymatic activity using the alternansucrase from Leuconostoc mesenteroides NRRL B-1355. Such a methodology might be used for glucan-synthesizing enzyme screening.120

Prospects

As illustrated above, the molecular evolution of glucansucrases has

shown to be powerful for modulating activities or creating new transglucosylation reactions (Fig. 5). Exploring further the fitness landscape of
Fitness
Variant

Variant
Sequence
Variant

WT

Sequence

Fig. 5 Exploring protein fitness landscape by directed evolution to modify specificity or

create new functions and modulating biophysical properties such as thermostability,
tolerance to solvents, flexibility. . .
640 | Carbohydr. Chem., 2014, 40, 624645

these enzymes may oer new opportunities for optimizing non-natural

functions and extend the range of synthesized products.121,122
Recent successes in the molecular evolution of glycoside-hydrolases
have paved the way to future investigations towards tailor-made oligosaccharides and glycoconjugates syntheses. Tools have been developed
for enhancing stability, activity, expression, promiscuity and specificity of
glucansucrases. Though ecient for transglucosylation reactions catalysis, the use of alternative donor substrates remains a major challenge
for glucansucrase applications. In this context, sucrose derivatives and
analogs appear relevant if utilized as substrates to further diversify the
glyco-structures generated by GS.27 Moreover, investigation of structureactivity relationships have to be deepened to further understand molecular determinants involved in enzyme stability and activity. Protein
dynamism exploration as well as detailed QM/MM studies may provide
pertinent perspectives in this purpose to adapt GS to non-natural substrates or analyze stabilizing eects.123125 Such information would be
crucial to further engineer variants with improved or novel properties of
interest and provide powerful catalysts for industrial applications.126

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