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Leloir Glycosyltransferases as Biocatalysts for Chemical Production Bernd Nidetzky, Alexander Gutmann, and Chao Zhong ACS Catal., Just Accepted Manuscript • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Catalysis

Leloir Glycosyltransferases as Biocatalysts for Chemical Production †, §, *

Bernd Nidetzky, †

Alexander Gutmann† and Chao Zhong

†

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology,

NAWI Graz, Petersgasse 12, A-8010 Graz, Austria §

Austrian Centre of Industrial Biotechnology (acib), Petersgasse 14, A-8010 Graz, Austria

ACS Paragon Plus Environment

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ABSTRACT

Glycosylation is a chemical transformation centrally important in all glycoscience and the related technologies. Catalysts offering good control over reactivity and selectivity in synthetic glycosylations are much sought after. The enzymes responsible for glycosylations in natural biosynthesis are sugar nucleotide-dependent (Leloir) glycosyltransferases. Discovery-oriented synthesis and pilot batch production of oligosaccharides and glycosylated natural products have previously relied on Leloir glycosyltransferases. However, despite their perceived synthetic utility, Leloir glycosyltransferases are yet to see widespread application in industrial biocatalysis. Here, we show progress and limitations in the development of Leloir glycosyltransferases into robust biocatalytic systems for use in glycosylations for chemical production. To obtain highly active and stable (whole cell) catalysts, able to promote the desired glycosylation(s) coupled to in situ sugar nucleotide supply, remains a difficult problem. To optimize glycosyltransferase cascade reactions for high process efficiency is another. Glycosylations of some natural products (e.g., flavonoids, terpenoids) involve acceptor substrate solubility as a special challenge for biocatalytic process design. Strategies to overcome these problems are illustrated from examples of integrated biocatalytic process development with this class of enzymes.

KEYWORDS: Leloir glycosyltransferases; sugar nucleotide; oligosaccharides; glycans; natural products; biocatalysis


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ACS Catalysis

1.

GLYCOSYLATION:

KEY

TO

CARBOHYDRATE

STRUCTURAL

AND

FUNCTIONAL DIVERSITY Glycosylation is the covalent coupling of sugar residues via glycosidic linkages. Glycosylation is 1

mainly responsible for the vast structural diversity of carbohydrates in nature. Glycosides, the products of glycosylation reaction(s), are molecules fundamentally important in biology, chemistry and the related technologies. Biologically, they include glycosylated natural products, 1-

oligo- and polysaccharides in free form or linked to other biomolecules like proteins and lipids. 4

Besides affecting the chemical properties (e.g., water solubility) of the product, glycosylation

gives rise to biological specificity and influences the mechanism of action.

5-7

A broad variety of

medicinally relevant molecules, including protein biopharmaceuticals, involve glycosylation(s) 8-10

in their (bio)synthesis.

In addition, glycosylated natural products and oligosaccharides have 5, 11

interesting uses as food and feed ingredients.

Furthermore, there is interest in small-molecule

glycosides as active components of cosmetic products and in flavor and fragrance 12-14

applications.

Scheme 1 shows selected examples of glycosylated products with promising

industrial applications. Development of robust methods of glycosylation is therefore a centrally important objective in the field. OH

OH

HO O HO

O

O

O

O HO

HO

O HO

OH O

O

HO HO

O

O

OH

HO

HO OH

geranyl O-β-D-glucoside

HN

2'-O-α-L-fucosyl-lactose

O

OH

HO OH

HO

O

O

O HO

HO

OH

OH

HO HO

OH

3'-O-α-sialyl-lactose

OH O OH

OH

HO HO

HO H

O HO OH

H

OH HO

O

O OH

HO

OH

OH

O

H

OH ginsenoside Rh2 protopanaxadiol 3-O-β-D-glucoside

HO HO

O O

HO HO

H O O

OH

O

O

OH O

HO O

OH OH

H HO

O

OH

quercetin 3-O-β-D-galactoside

HO HO

H

O O

rebaudioside A

Scheme 1. Industrially relevant oligosaccharides and glycosides referred to in this review.


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15-19

Chemical methods,

2-3, 20-21

biocatalytic approaches

Page 4 of 45

4, 22-23

and combinations thereof

have

been considered for glycosylation. Controlling selectivity and reactivity is a difficult problem for glycosylation. For production-scale glycoside synthesis, green chemistry characteristics of the 24

process require particular attention.

Prevention of waste (e.g., through avoiding protection

group chemistry) and use of catalysis are important principles to be considered. Enzymes are promising as catalysts for glycoside synthesis. However, there exists a significant gap in scope between the glycosides in demand and the enzyme-catalyzed transformations available for their synthesis. Development of new enzymes for glycosylation applications is therefore important.

2. LELOIR GLYCOSYLTRANSFERASES 3-4, 25

The natural principle of glycoside synthesis is represented by Leloir glycosyltransferases.

These enzymes utilize activated sugar donors, typically sugar nucleotides, as substrates. Chemically, glycosyltransferase reactions are nucleophilic substitutions at the glycosyl anomeric carbon and proceed with inversion or retention of configuration,

1-2, 25

as shown in Scheme 2.

Scheme 2. Inverting (A) and retaining (B) glycosyltransferase reactions are shown. Net proton release occurs in glycoside synthesis at pH conditions (pH ≥ ∼7), in which the nucleoside diphosphate is unprotonated.

1, 26-27

Glycosyltransferases are stereoselective enzymes. In reactions with acceptors offering several 4, 11,

glycosylation sites (see Scheme 1), glycosyltransferases usually show good site selectivity. 13-14, 25, 28

Glycosyltransferases have therefore been considered potential "game changing"


4

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ACS Catalysis

catalysts of glycosylation, enabling high-precision glycoside synthesis in single-step conversions.

11, 13, 28

Iterative glycosylations are possible without the need of intermediary

28-32

product purification.

However, despite these advantages, glycosyltransferases are yet to play

a significant role in industrial glycoside production. For synthesis of fine or specialty chemicals, glycosyltransferases are often considered rather difficult to use.

11

There are excellent reviews on glycosyltransferases, focusing on their structure, function and mechanism

3-4, 25, 28, 33-39

11, 13-14, 30, 40-41

6,

as well as on the principles and scope of their synthetic applications.

However, glycosyltransferases in biocatalytic processes have received little

attention. This has motivated the current study.

2.1 Basic attributes of Leloir glycosyltransferases. Glycosyltransferases have been classified based on sequence similarity. There are currently 105 glycosyltransferase families, comprising about 400,000 enzyme modules categorized in the CAZY (carbohydrate-active enzymes) 25, 42

database.

Contrasting their diversity in function, glycosyltransferases adopt only a few basic

structural folds, of which the GT-A and the GT-B fold are most common (Figure 1). GT-A type 2+

2+

glycosyltransferases often require a divalent metal ion (Mg , Mn ) for activity. These features of glycosyltransferases have been reviewed elsewhere.

25, 37, 43-44

Figure 1. Structures of glycosyltransferases of GT-A (A, galactosyltransferase LgtC from Neisseria meningitidis, PDB code: 1G9R) and GT-B fold (B, flavonoid glucosyltransferase from Vitis vinifera, PDB: code: 2C9Z; C, macrolide glycosyltransferase OleD from Streptomyces antibioticus, PDB code: 2IYF) used in biocatalysis and referred to in this review.


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25, 33,

Leloir glycosyltransferases catalyze glycosylation at O, N, S and aromatic C acceptor sites. 37-38, 45 37-38, 46

33,

They usually do so with good discrimination between different chemical sites present.

Mechanistically, as shown in Scheme 3, the inverting glycosyltransferases employ a 25, 33, 36

single displacement-like reaction, usually under catalytic assistance from a general base.

Aromatic C-glycosylation with inversion requires a slightly different mechanism (Scheme 3). Deprotonation of a suitably positioned, ortho or para hydroxy group may be used to generate 38, 46-50

nucleophilic character in the reactive carbon of the acceptor substrate.

Glycosyl transfer 25,

with retention appears to proceed in a single step without formation of a covalent intermediate. 36

However, covalent catalysis may be utilized by certain glycosyltransferases.

36

In terms of substrate selectivity, Leloir glycosyltransferases cover a broad range of donors and 11, 13, 33, 40

acceptors.

Individual enzymes can be anything from highly specific for a single pair of

donor and acceptor substrates to showing broad tolerance to structural variations in both. Some 51-52

glycosyltransferases promote multiple glycosylations on a single acceptor molecule 31-32

elongate a growing glycan chain.

or

These enzymes have proven useful for synthesis of

diglycosylated natural products and glycosaminoglycan oligosaccharides.

11, 31-32

2.2 Characteristics of the glycosyltransferase reaction. In glycosyltransferase reactions, both 25, 36

substrates must bind to the enzyme to form a ternary complex before conversion takes place.

Substrate binding and product release may involve a certain order, as for example in human α1,4-galactosyltransferase, in which binding of the sugar nucleotide is required for binding of 53

the acceptor.

However, in various other cases, substrate binding was random.

54

Binding of one

substrate usually enhances the affinity of the other substrate. For practicality of the enzyme, its specific activity, expressed in the catalytic constant (kcat), is important. Compared with glycoside hydrolases/transglycosidases (e.g., esterases/lipases,

57

55-56

and other enzymes having established biocatalytic applications 58

alcohol dehydrogenases ), glycosyltransferases often exhibit a rather 59

low kcat. Exceptions notwithstanding,

-1

typical kcat values are in the range 1 - 20 s or less.

60-63

For practical considerations, the Km value should be low relative to the substrate concentration used in synthesis. However, substrate inhibition is often connected to a low Km.

64


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ACS Catalysis

Scheme 3. Proposed mechanisms for glycosyltransferases.

25, 33, 36, 34

glycosyltransferase; B, inverting aromatic C-glycosyltransferase;

A, inverting O, N or S-

38, 46 41-45

C, retaining

glycosyltransferase acting through a concerted (top) or ion-pair intermediate (bottom) front-face mechanism; D, retaining glycosyltransferase forming a transient covalent intermediate.


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Page 8 of 45

61, 65-69

The nucleoside diphosphate released in the reaction may cause product inhibition.

Binding of acceptor to the enzyme-nucleoside diphosphate complex potentially augments the inhibition. The nucleoside diphosphate also affects the conversion by mass action. 70

Glycosyltransferase reactions are not practically irreversible,

contrary to what was long

thought. The nucleoside diphosphate may be hydrolyzed in situ using phosphatase.

30, 71

This

eliminates the inhibition and pulls the reaction to the product side. In situ recycling of sugar nucleotide from nucleoside diphosphate presents an alternative strategy to cope with issues of inhibition and reaction equilibrium (see later). The reaction equilibrium, with common Keq values of 10 or higher, typically favors glycoside synthesis.

1, 25

Depending on the difference in pKa between the glycosyl acceptor group (e.g., ∼12

- 14 for a sugar hydroxy group) and the terminal phosphate of the nucleoside diphosphate (∼ 6 7), the reaction equilibrium is pH dependent (Scheme 2). The Keq increases with increasing pH 1, 26, 72

in the relevant pH range (e.g., 6 - 12).

The glycosyltransferase reaction will thus involve a

net proton release (pH ≥ pKa of nucleoside diphosphate), which has previously been exploited for development of enzyme assays suitable for high-throughput experiments. Glycosyltransferase reactions may require pH control.

27, 73-74

26

Stability problems confront glycosyltransferase synthesis in different ways. Enzymes often show half-lives of just a few hours under reaction conditions.

61, 75-77

The total turnover number

(TTN) for the enzyme thus becomes an issue. For a small-molecule glycoside, therefore, a TTN 6

of 10 enables production of several kg product/g enzyme used. TTN values are not widely 4

5 75, 78-79

available for glycosyltransferases, but seem to be relatively low (10 - 10 ) to industrially used enzymes for carbohydrates transformations.

80-81

in comparison

More research into stability

and stabilization of glycosyltransferases is needed. Sugar nucleotides can decompose spontaneously within times (24 - 48 h) relevant for the 82-86

2+

2+

enzymatic synthesis.

Besides pH and temperature, metal ions (e.g., Mg , Mn ) also affect

82-83, 86

For example, uridine 5'-diphosphate α-D-glucose decomposes by releasing

their stability.

83,

uridine 5'-monophosphate under formation of α-D-glucose 1,2-cyclic phosphate.

86


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ACS Catalysis

Establishing conditions that prevent donor substrate degradation thus becomes a relevant task in reaction optimization.

2.3 The glycosyltransferase reaction in synthetic context. Glycosyltransferase synthesis is a field in which biocatalysis becomes strongly intertwined with synthetic biology and metabolic 2, 6, 30, 87-91

engineering.

The donor and acceptor substrates for the enzymatic reactions are often

not available as reagents. Their preparation from expedient precursors involves multistep 30, 89, 92

(chemo)-enzymatic reaction cascades.

These cascades are realized as "self-sufficient"

bio-synthetic pathways in vitro or through integration with cellular metabolism. For in vitro synthesis, isolated enzymes or whole cell(s) might be used. Live cell factories enable glycosides to be synthesized from nutrients. Enzyme- and cell-based glycosylations represent complementary strategies of glycoside production. Their comparison as to the limits of efficiency is of interest, but not available to our knowledge. Here, we focus mainly on enzymatic cascades uncoupled from metabolism. However, the points discussed are in the main also applicable to glycosylation by live cells. The main, and by far most important, strategy of applying glycosyltransferases in biocatalysis is synthesis of glycosides as final products. Installing glycosyl residues as biochemical protecting groups, which are removable on demand, is an alternative application of glycosyltransferases (Scheme 4). Indeed, glycosylation is used naturally for detoxification

93-94

and as "self-

7

resistance" mechanism.

Scheme 4. Glycosylation as a protecting group strategy for biotechnological production (left, middle) and for chiral resolution applications (right) is shown.


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Page 10 of 45

The toxicity of vanillin to yeast cells is decreased through β-D-glucoside formation during in 95

vivo production. Indoxyl, which is a highly reactive precursor of the common blue dye indigo, was glucosylated by a dedicated β-glucosyltransferase to form the comparably unreactive and much

better

soluble

derivative,

96

called

indican.

Another

possible

application

of

glycosyltransferases is resolution of racemic substrates through enantioselective glycosylation. The enzyme UGT71B6 from Arabidopsis thaliana was highly selective for reaction with (+)abscisic acid to form the corresponding β-glucosyl ester, thus enabling the natural (+) form to be 40

resolved from the (±) substrate (Scheme 4).

The reader may note that biocatalysis offers 97

various strategies for chiral resolution and also for introducing protecting groups. Glycosylation presents only one among a number of possibilities.

3. GLYCOSYLTRANSFERASES AS BIOCATALYSTS Glycosyltransferases are notorious for their difficult preparation as biocatalysts. The problem requires attention, because efficiency in biocatalyst preparation could compensate deficiencies of a glycosyltransferase as regards specific activity and stability. Besides selection of host and genetic systems, design of the protein for soluble expression proved important with many glycosyltransferases (e.g., targeted truncation to remove membrane-anchoring parts; fusion to 98-100

solubility enhancer modules; removal or substitution of aggregation prone elements).

Overall, microbial glycosyltransferases are more easily produced recombinantly than their relatives from higher organisms. They are therefore considered most promising for biocatalytic 28, 30, 37, 101

applications.

It is difficult to give a general account and we use representative

examples instead. Tables S1 and S2 (Supporting Information) summarize expression data for bacterial sialyltransferases and fucosyltransferases produced in E. coli. Large variation in protein and activity yield is noted depending on the enzyme and expression strategy used. Volumetric 3

-1

activities for the best expressing sialyltransferases are at around 1 × 10 units L . This is significantly less than activities reached with other enzymes (e.g., transglycosidases, glycoside phosphorylases) used for glycosylation. To give an example, recombinant production of sucrose 3

-1

phosphorylase (from Leuconostoc mesenteroides) in E. coli yielded about 600 × 10 units L of


10

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ACS Catalysis

81

bioreactor culture.

This emphasizes the important leverage for the biocatalytic applications of

glycosyltransferases that would be achievable through efficiency-enhanced production of the enzymes. Besides boosting the volumetric activity, the specific activity (based on cell mass) might be enhanced. This in turn would immediately benefit the use of glycosyltransferases as whole cell biocatalysts, rendering enzyme isolation a process step no longer necessary. In addition to microbial glycosyltransferases, glycosyltransferases from higher organisms are of interest for synthesis. For example, protein glycan remodeling may be achieved effectively 102

using mammalian glycosyltransferases (e.g., sialyltransferase

). Plant glycosyltransferases

have unique specificities for glycosylating "their own" natural products, including a broad diversity of flavonoid and terpenoid acceptor molecules.

13-14, 34, 40, 60

Besides E. coli, which is

nearly always considered as expression host, platform systems for high-throughput expression of mammalian glycosyltransferases are mammalian cells (e.g., HEK293) and insect cells. 104

free systems seem to be gaining in importance.

103

Cell-

Yeast systems may not be more suitable than 105-

E. coli for the functional overexpression of either mammalian or plant glycosyltransferases. 106

4.

GLYCOSYLTRANSFERASE-CATALYZED

SYNTHESIS:

SYSTEMS

AND

PARAMETERS FOR PRACTICAL USE Large-scale synthesis of oligosaccharides and glycoconjugates was performed directly from the sugar nucleotide (e.g., UDP-Gal, UDP-GlcNAc) and the corresponding acceptor.

107-109

However, sugar nucleotides should be supplied more efficiently than by simply adding them. Sugar nucleotide formation in situ is therefore useful.

6, 21, 30, 110

52, 111-112

supply apply similarly to the acceptor molecule.

Consideration of in situ substrate

This was however outside of the scope

of the current review. 4.1 Enzymatic cascades for sugar nucleotide supply. Scheme 5 summarizes strategies to prepare sugar nucleotides from intermediary sugar phosphates. Phosphorylation at the anomeric center provides the greatest flexibility as result of discovery and engineering of anomeric kinases with 21, 30, 113-114

different substrate specificities.

Stereoselective anomeric phosphorylation of sugars


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Page 12 of 45

was also achieved by a phosphatase-catalyzed transphosphorylation using α-glucose 1-phosphate as phosphate donor. 113

activity.

115

There is paucity of anomeric kinases phosphorylating Glc with high

Besides hexokinase and phosphoglucomutase, glycoside phosphorylases can be used

to release α-glucose 1-phosphate. Sucrose is the most convenient phosphorylase donor substrate.

116

Sugar-1-phosphate synthesis α-Glc-1P

NDP-sugar synthesis Glc

IPP

Pase

O HO

O

HO

OH

AK

sugar

OH

OPO32-

ATP

ADP

ATP

ADP HK

HO

HO OPO 32-

OPO32-

HO

OH

OH O

HO HO

PGM

f ru

HO HO

Pi

O

a SP

HO

HO HO

OPO3

se

O sucrose

O

+ HO OH

2-

HO

HO

α-Glc-1-phosphate

O HO

HO

OPO32-

O UDP UDP-Gal

α-Gal-1-phosphate

OH

HO

GalT

OH O

HO

OPO32-

α-Glc-1-phosphate

+

O

HO

O UDP

UDP-Glc

OH

O

HO HO

HO

Glc-6-phosphate

OH

O NDP NDP-sugar

OH

O

Glc

se cto

O

NTase

sugar-1-phosphate

HO HO

OH

PPi

HO

sugar-1-phosphate

O

HO HO

NTP

O

2 Pi

ase

OH OH

Glycosylation

OH

O

NTP regeneration

+

HO

pyruvate

HR

PEP

R = O, N, C, S

acceptor

O NDP NDP-sugar

PK

GT PolyPn NTP

PolyPn+1 NDP

PolyPK

O acetate

acetylphosphate

NDP

AcetateK

+

O

HO glycosylated acceptor

Scheme 5. Cascade reactions for sugar nucleotide supply to glycosyltransferase conversions. AK, anomeric kinase; Pase, phosphatase; HK, hexokinase; SPase, sucrose phosphorylase; Pi, inorganic phosphate; NTase, pyrophosphorylase; PPi, pyrophosphate; IPPase, inorganic


12

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ACS Catalysis

pyrophosphatase; GalT, hexose 1-phosphate uridylyltransferase; GT, glycosyltransferase; PK, pyruvate kinase; PEP, phosphoenolpyruvate; PolyPK, polyphosphate kinase; PolyPn, polyphosphate with n phosphate groups; AcetateK, acetate kinase.

Various sugar nucleotide pyrophosphorylases have been used for conversion of sugar 16, 30

phosphate into the corresponding sugar nucleotide.

Pyrophosphatase is added to drive the

reaction and to remove product inhibition by the pyrophosphate released. UDP-Glc hexose 1phosphate uridylyltransferase catalyzes the interchange between UDP-Glc and sugar 1phosphates (e.g., α-galactose 1-phosphate). Lacking a suitable pyrophosphorylase, this enzyme can be used to form UDP-Gal.

4, 117

The nucleoside triphosphates required in these reactions are

normally regenerated. The pyruvate kinase/phosphoenolpyruvate system is well established. Polyphosphate kinase was considered as an alternative, for polyphosphate would be a highly expedient phosphate donor. However, polyphosphate reactions (e.g., complexation, precipitation) with divalent metals are difficult to control and removal of residual polyphosphate from reaction product is an issue.

118

Acetate kinase/acetylphosphate has also been proposed for nucleoside 51

triphosphate recycling.

Stability of acetylphosphate over longer reaction times might be a

problem. The donor substrate of sialyltransferases, CMP-Neu5Ac, requires a unique enzymatic cascade for its in situ formation.

4, 30, 89, 119-120

This is shown in Scheme 6. NH2

O

OH HO HO

GlcNAc

NH O

pyruvate

OH

O

HN

AGE OH

HO HO

NAL

O

OH HO HN

OH O

ManNAc

O

O OH

NH

CTP

OH

OH

CSS

O

Neu5Ac

HO PPi

O N O P O O OH O OH O OH OH O HN O OH O

CMP-Neu5Ac

Scheme 6. Cascade reactions for sialic acid donor supply to sialyltransferase conversions. AGE, GlcNAc 2-epimerase; NAL, sialic acid aldolase; CSS, CMP Neu5Ac synthetase

2-Epimerisation of GlcNAc is an interesting extension of the cascade originally established from ManNAc or Neu5Ac.

119-121

Additional routes towards Neu5Ac (e.g., sialic acid


13

O

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122

synthase

123

; hydrolyzing UDP-GlcNAc epimerase

Page 14 of 45

) might be employed in live cell factories

and the reader is referred to the cited literature for their in-depth discussion. 4.2 Glycosyltransferase reactions run in reverse direction for sugar nucleotide supply. The fundamental insight that, through proper choice of reaction conditions 125

activated, non-natural glycosidic substrate,

70, 124

or use of a highly

glycosyltransferase reactions can be driven in their

reverse direction has found immediate practical interest. Sucrose synthase is a rare example of a Leloir glycosyltransferase whose natural reaction, sucrose + nucleoside diphosphate (NDP) ↔ NDP-glucose + fructose, has an equilibrium lying 126

plainly in the middle.

The reaction presents a powerful method of glucose nucleotide 61

synthesis using just a single enzyme.

Its utility for sugar nucleotide supply was extended

through coupling to further enzymatic step(s). UDP-Gal, UDP-GlcA and dTDP-rhamnose were thus prepared from UDP-glucose and utilized immediately in coupled glycosyltransferase 61

reactions.

synthesis.

Like sucrose synthase, trehalose synthase was used for glucose nucleotide

127

However, the glycosidic bond in α,α-trehalose is of much lower free energy than 128-129

that of sucrose.

This renders α,α-trehalose less attractive than sucrose as donor substrate 127

for NDP-glucose production. Although some efforts were made,

coupling of trehalose

synthase with other glycosyltransferase requires further study to demonstrate its synthetic utility. Seminal studies of Thorson

125

and Withers

130

revealed that simple glycosides having aromatic

leaving groups can function as donor substrates for the reverse reaction of glycosyltransferases. 125

Gantt et al.

showed that through structural variation in the aromatic leaving group of the β-

glycosidic substrate, glycosyltransferase conversions into sugar nucleotide product could be shifted from a process disfavored thermodynamically to one strongly favored. Examining a quadruple variant (TDP-16) of the macrolide-inactivating glycosyltransferase OleD (from Streptomyces antibioticus) in the presence of 2-chloro-4-nitrophenyl glycoside donors (Scheme 7A), a panel of 22 sugar nucleotides was synthesized. This panel featured broad diversity in both the sugar and the nucleoside diphosphate part of the molecule. Ability of the OleD variant to synthesize different sugar nucleotides was exploited in one-pot transglycosylations wherein the sugar nucleotide released from the activated glycoside served as donor for the subsequent


14

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ACS Catalysis

glycoside-forming reaction. The generic principles revealed by Gantt et al. have since been applied in many studies of glycosyltransferase specificity and small-scale glycoside synthesis by 131-138

They have also proven extremely useful in enzyme discovery and in

these enzymes.

diversifying the glycosylation pattern of natural products, applying glycosyltransferases as isolated enzymes

139

or as whole-cell catalysts.

140

A NO2

O HO HO

O

O

Cl

O

OH

XH

OH

NDP

OH

HO HO

OH

NO2

HO HO

OH O NDP

X

OH

NDP X = O, N, S, C

HO

Cl Recycle

B OH HO

OH O-GT

OH

OH

O HO HO

O

O

OH

HO

OH

OH

phlorizin

UDP

HO HO

O OH

HO O

OH

UDP

O phloretin

OH HO O HO HO

HO

OH C-GT OH

O

nothofagin

Scheme 7. Glycosyltransferase (aglycon) exchange reaction pushed by an activated sugar donor (A) and O-C glycosidic bond rearrangement pulled by the relatively higher stability of the Cglycosidic product (B). Both reactions require only catalytic amounts of NDP. They involve two complementary glycosyltransferase activities from a suitable pair of enzymes or from a single promiscuous enzyme. The reaction in A can be adapted for exchange of sugar instead of aglycon.

Based on Thorson's work of glycosyltransferase reversibility,

64

Liu and colleagues used kinetic 141

simulations to analyze sugar/aglycon exchange reactions by coupled glycosyltransferases.

They concluded that Keq of the individual transfer reactions governs the outcome of the overall


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Page 16 of 45

transglycosylation. The equilibria of sugar/aglycone exchange reactions and their role for practicable synthesis thus require more detailed attention. The additional importance of kinetic characteristics of the enzyme(s) used was revealed in a study of O- to C-glycoside rearrangement 72

by coupled glycosyltransferases (Scheme 7B).

Although strongly favored thermodynamically,

conversion of phloretin 2'-O-β-D-glucoside (phloridzin) into phloretin 3'-C-β-D-glucoside (nothofagin) proceeded efficiently only in the case that kinetic fluxes to and from UDP-glucose 72

were suitably balanced.

Certain C-glycosyltransferases catalyze (reversible) O-glycosylation 50, 134, 142

on surrogate substrates in vitro.

This ability might be exploited in further applications

of O- to C-glycoside rearrangement using only a single enzyme.

142

4.3 Synthetic applications of glycosyltransferases using sugar nucleotide formation in situ. Representative examples of synthetic applications of glycosyltransferases in biocatalytic cascades are given. Besides conceptual relevance, demonstrated scalability was especially taken into account. The different glycosylation systems used are paramount examples of cascade biocatalysis applied to chemical synthesis. However, with exceptions,

143

the almost complete

lack of engineering principles (e.g., kinetic model-based optimization; reaction/reactor design) involved in their development is noted. 4.3.1

Oligosaccharides.

The

synthetic

scope

of

oligosaccharide

synthesis

by

glycosyltransferases and biocatalytic glycosylations by one-pot multienzyme systems are 11, 21, 30

covered well in earlier reviews. 144

sialyllactose,

147-148

3-lactose)

Large-scale synthesis of oligosaccharides, like 3'144-146

globotriose (Gal-α1-4-lactose)

and the α-galactosyl trisaccharide (Gal-α1-

, by engineered whole cell catalysts remain important points of reference. -1

Although product titers of up to 188 g L (372 mM; 2.5 L scale) were obtained in often excellent yields, the cell loading applied to these conversions was huge, typically in the range 100 - 250 g -1

144

L . Biocatalysts were multiple bacterial species

or a single "superbug".

91, 145-149

A large

benefit is expected from improved overexpression of the enzymes, the glycosyltransferases in particular, in the respective host organism. Balanced co-expression of enzymes could be an additional problem. It might be addressed with modern methods of synthetic biology.

91, 150-151


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ACS Catalysis

Living cell factories, often constructed in E. coli strain background, present alternative -1

production systems for oligosaccharides, including 2'-fucosyllactose (23 g L ), 3'-sialyllactose -1

-1 87-88, 90, 152

(25 g L ) and 6'-sialyllactose (34 g L ).

Intracellular accumulation of a significant

portion of product (30 - 40%) is a problem for downstream processing. However, the synthesis of 2'-L-fucosyllactose is now moving towards full-scale commercialized production in industrial processes.

87

This occurs under product approval for application as food ingredient from the

relevant regulatory agencies in the U.S. and the EU. Their inherently flexible use renders cell-free enzyme preparations the preferred choice for diversity-oriented synthesis, typically for production of up to the gram scale. Here, the 100-g scale production of 3'-sialyllactose (Scheme 1) from Neu5Ac and lactose by a fusion protein between CMP-sialic acid synthetase and α2-3 sialyltransferase (both from Neisseria 153

meningitidis) is a key point of reference. 30

nucleotide regeneration (Scheme 5),

By applying one-pot multienzyme cascades for sugar

more recent studies demonstrated impressively the use of

sequential glycosylations for the production of all 15 naturally occurring human ABH antigens;

154

a large panel of complex human milk oligosaccharides;

Lewis X antigens; sphingolipids;

158

157

and 159-160

oligosaccharides.

155-156

O-sulfated sialyl

a pentasaccharyl ceramide representing a class of complex structurally

well-defined

chondroitin

sulfate

and

heparosan

The efficiency of sugar nucleotide regeneration (UDP-Gal, UDP-

GalNAc, GDP-Fuc, CMP-Neu5Ac) was evaluated in detail for gram scale syntheses of the cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 and sialyl-Gb5, which were 78

assembled from allyl-lactose (55 mM) through sequential enzymatic glycosylations.

The mole-

based TTN for the enzymes used in a single batch reaction (2 - 10 days; 150 - 200 mL) without 4

6

enzyme recycling was in the range 10 - 10 . For nucleoside triphosphates, TTN values were between 200 (ATP) and 20 (CTP, GTP). Isolated yields for intermediary and final products were high (94 - 99%), except for sialyl-Gb5 (54%). To enhance the TTN for the enzymes, their coimmobilization on beads could be useful, as was shown for UDP-Gal regeneration cascades. 162

161-

Enzymes were immobilized via their His-tags on nickel-nitrilotriacetate beads with excellent

retention of activity on solid as compared to solution. Reuse of the beads for UDP-Gal


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Page 18 of 45

production (10 mM; 48 h; 50% yield) was possible at least four times. Oligosaccharide syntheses with α1-3 or α1-4 galactosyltransferase, or both enzymes, immobilized on beads that additionally contained the UDP-Gal regeneration cascade gave product yields of between 76 and 95%, based on 60 mM substrates used. In a gram scale production of a β-benzyl derivative of the α-Gal trisaccharide, 3-fold reuse of the beads was demonstrated with only small losses of product yield in each round of conversion (72%, 71%, 69%, 66%).

162

Protein engineering was used to adapt glycosyltransferases to requirements of oligosaccharide 163

synthesis. Bacterial sialyltransferases showing reduced hydrolytic activity 164-165

acceptor site-selectivity

and changed

proved useful. Ultra-high-throughput screening combined with

directed evolution was used to develop variants of Campylobacter jejunii sialyltransferase CstII 166

with enhanced efficiency.

The α1-3 fucosyltransferase (from Helicobacter pylori) was -1

systematically truncated at the C-terminus to improve solubility (150 - 200 mg of ∆52 variant L

E. coli culture). Structure-guided iterative saturation mutagenesis was used to enhance activity (15.5-fold compared to wildtype enzyme).

99

4.3.2 Natural product glycosides. Glycosyltransferase biocatalysis for natural product glycosylation has attracted significant interest. Microbial enzymes and enzymes from plants have been studied extensively, with a strong focus on enzyme discovery and mining for new enzyme 13-14, 34, 37, 40, 167-171

specificities.

Due to the huge diversity of aglycon structures in natural

product glycosides, a broad-scope toolbox of versatile glycosylation catalysts is desired. Several of these glycosyltransferases are unexpectedly promiscuous, particularly regarding the acceptor 40, 131-135

molecules utilized.

This property is potentially very useful for biocatalytic synthesis.

Despite progress in elucidating structures of natural product glycosyltransferases, correlating sequence and structure to function is still difficult.

25, 33, 37

Bioprospecting from genomes of 33

glycosyltransferases with assignable specificities therefore remains a distant goal.

Sequence-

guided or structure-based design has had some successes in glycosyltransferase engineering for 37, 43, 167

biocatalytic applications.

However, induced-fit substrate binding is a structural53, 172

mechanistic feature apparently quite common among the glycosyltransferases.

Rational

engineering should therefore rely on structural evidence of the ternary Michaelis complex or a


18

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ACS Catalysis

173-174

Besides swapping sequence regions or whole domains between

good surrogate thereof.

47, 74, 175-177

different glycosyltransferases,

37, 167

of new enzyme specificities.

directed evolution proved useful in the development

It is noted that good protein expression is a critical

requirement for directed evolution to take its full effect in creating the desired trait(s) in an enzyme. Advances in high-throughput glycosyltransferase assays have supported enzyme 74, 166, 178-180

directed evolution.

Glycosyltransferase engineering for promiscuity and for a specific function. The substrate scope of OleD was expanded dramatically through a directed evolution combined with high37, 178, 181

throughput fluorescence screening (Scheme 8A).

The triple variant (Pro67Thr

Ser132Phe Ala242Val) utilized 15 donor substrates of which 12 were "new". It was superpromiscuous with respect to the acceptor substrate, leading to ≥ 50 "new" candidate molecules for glycosylation. Furthermore, the variant was able to glycosylate different nucleophiles, including alcohols, amines, thiols, oximes, hydrazines, hydrazides, N-hydroxyamides, Osubstituted oxyamines and carboxylic acids. Further engineering of OleD improved the enzyme's efficiency for sugar nucleotide synthesis. Quintuple variant incorporating additional substitutions Ile112Pro and Thr113Met showed >400-fold improvements in kcat/Km for formation of different NDP-(deoxy)-glucoses.

182

The enzyme was immobilized through His-tag affinity binding. Its

use under flow conditions afforded a 280-fold improved enzyme utilization as compared to homogeneous reaction using the soluble enzyme.

183

Engineering of a glycosyltransferase for a defined target function is represented by UGT51 (from Saccharomyces cerevisiae).

179

The enzyme whose natural function is believed to be the

synthesis of ergosteryl β-D-glucoside is inherently promiscuous for β-D-glucosylation of sterols. To enable production of the ginsenoside Rh2 (Scheme 1), UGT51 was engineered for enhanced reactivity with the precursor sterol protopanaxadiol. Screening according to Scheme 8B was performed. The best improved 7-fold variant (Ser81Ala Leu82Ala Val84Ala Lys92Ala Glu96Lys

Ser129Ala

Asn172Asp)

showed

1800-fold

enhanced

efficiency

towards

-1

protopanaxadiol, with a kcat of 1.4 s and a Km of 0.28 mM. The engineered glycosyltransferase was useful to boost ginsenoside Rh2 production in recombinant Saccharomyces cerevisiae.

184


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Page 20 of 45

OH

B

A

HO GT (variant)

O HO

O

glucosylated acceptor

UDP

HO O UDP

HO

UDP-glucose

GT 2

O HO

O

OH O

O

4-methyl-umbelliferone

4-methyl-umbelliferyl glycoside

fluorescent

non-fluorescent

O

Cl

O

O

HO HO

Cl O

OH NO2

NO2 2-chloro-4-nitrophenolate

yellow

2-chloro-4-nitrophenyl β-D-glucopyranoside

colorless

Scheme 8. High-throughput glycosyltransferase reactions with fluorescence (A) or absorbance (B) detection are shown. They were used in enzyme engineering.

185

In another study of targeted glycosyltransferase engineering,

the acceptor binding pocket of

the enzyme UGT76G1 (from Stevia rebaudiana) was subjected to extensive site-saturation mutagenesis at 38 positions. UGT76G1 participates in a complex metabolic glycosylation grid that leads to numerous glyco-forms of steviol, some of which are desired as sweeteners like rebaudioside D and rebaudioside M whereas others like stevioside and rebaudioside A (Scheme 1) are undesired for their additional bitter taste. UGT76G1 catalyzes β1-3 glucosylation of glucosyl residues attached to the steviol core molecule. Variant enzymes (Thr146Leu or His115Leu) showing improved participation in the network of glycosylation reactions and consequently reduced by-product formation in vivo (S. cerevisiae) were identified. An important finding from structure-function studies of the mannosyl glycerate synthase (from 186

Rhodotermus marinus)

O

O

HO HO

NDP-sugar

GT (variant)

HO

OH

NDP

O NDP

O

HO HO

acceptor

was that mutation of residues interacting with the guanine base of

GDP were correlated with large increases in kcat at relatively unchanged kcat/Km. If this were a principle broadly applicable to glycosyltransferases, one could foresee interesting practical applications in the design of rate-enhanced glycosylation catalysts for synthetic use.


20

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ACS Catalysis

Application of natural product glycosyltransferases in biocatalytic synthesis. The enormous structural diversity of glycosylated natural products necessitates discussion based on 13-14, 34,

representative examples. Excluding discovery- and mainly diversity-oriented synthesis, 40, 51, 60, 168, 177, 187

we focus on simple glycosylations (e.g., glucosylation, galactosylation) of

flavonoid and terpenoid scaffolds (Scheme 1). This is in consideration of the various possible applications that the resulting glycosidic products have as industrial fine chemicals and 12-14, 188

ingredients.

Acceptor substrate solubility is a problem largely absent from biocatalytic glycosylations of sugar molecules, but one that requires special attention in the glycosylation of almost any natural product. Generally, therefore, product titers are severely limited by the amount of acceptor 51-52, 60

solubilized (usually ≤ 1 mM).

Substrate solubility enhancement is certainly not new to 24

biocatalysis and various well-known strategies have been developed to its end,

yet, it must be

noted, they all necessitate a (highly) robust enzyme. The simplest approach involves a whole-cell catalyst prepared from the host organism, most often E. coli, overexpressing the relevant glycosyltransferase. The sugar nucleotide is provided by (residual) cellular metabolism on addition of Glc or Gal to the reaction mixture. Typical -1 40, 52

product concentrations were in the approximate range 20 - 200 mg L .

Using a water

immiscible ionic liquid, N-hexylpyridinium bis(trifluoromethylsulfonyl)imid, as a second phase for fed-batch supply of the geraniol acceptor substrate,

189

geranyl β-D-glucoside (Scheme 1) was

-1

-

produced in 20 h to a concentration of 291 mg L by a whole-cell catalyst (4.3 g cell dry mass L 1

) based on E. coli BL21(DE3) expressing the Arabidopsis thaliana glucosyltransferase GT14a. -1

The reference reaction used geraniol dissolved in buffer and gave 118 mg product L . Recently, isopropyl myristate was shown to be a promising organic solvent for geranyl β-D-glucoside 190

production.

In a different study, a Saccharomyces cerevisiae strain expressing the UGT76G1 -1

(from Stevia rebaudiana) was used as whole cell catalyst to produce 1.2 g L rebaudioside A -1 191

(Scheme 1) from stevioside (2 g L ).

Besides glucose, citrate was added to enhance the

accumulation of intracellular UDP-glucose from the carbon source.


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Page 22 of 45

Strains harboring engineered pathways of donor substrate supply showed enhanced production -1 52

-1

or even ∼4 g L in the

of flavonoid glycosides with titers raised to several hundred mg L case of quercetin 3-O-β-D-glucoside.

192

The scope of sugars used for glycosylation was

broadened to include, among others, Xyl, Rha, GlcA, and L-Ara, which occur naturally in known 51-52, 193

flavonoid glycosides.

Besides UDP-sugars, TDP-activated sugars were used. Multiple

glycosylations on a single acceptor were also performed. De Mey and colleagues

194

constructed a

pathway in E. coli wherein UDP-Glc was synthesized from sucrose via α-glucose 1-phosphate. The UDP-Glc was the immediate precursor for UDP-Gal (Bifidobacterium bifidum UDP-glucose C'4 epimerase) and UDP-Rha (Arabidopsis thaliana UDP-Rha synthase; MUM4 gene) utilized for glycosylation of quercetin. A flavonol 3-O-glacatosyltransferase (from Petunia hybrida) or flavonol-3-O-rhamnosyltransferase (from A. thaliana) was overexpressed. In 1-L bioreactor -1

cultivations quercetin 3-O-galactoside (hyperoside, Scheme 1; 0.94 g L ) and quercetin 3-O-1

rhamnoside (quercetrin; 1.15 g L ) were formed within 30 h in a yield of roughly 50% based on the quercetin added. Using a similar approach employing a versatile glucosyltransferase (from Vinis vitivera), a series of carboxylic acids were converted to the corresponding β-glucose esters.

195

Other interesting examples are the glucosylation of hydroquinone to give arbutin β-D-1 196

glucoside (6.62 g L )

-1 197

and of salicylate to give salicylate 2-O-β-D-glucoside (2.5 g L ).

Enzymatic glycosylations were performed on various acceptors (e.g., quercetin, resveratrol, phloretin, stevioside, curcumin) using sugar donor regeneration.

60, 198-208

However, final product

concentrations, after some optimizations, were typically still in the low mM range, reflecting limitations on acceptor substrate solubility. Unlike oligosaccharide synthesis, therefore, these reactions can hardly challenge the performance of the regeneration systems used. Intensification of enzymatic flavonoid glycosylation with in situ sugar nucleotide recycling is described below based on the example of synthesis of the C-glycoside nothofagin. Sugar or aglycon exchange reactions were exploited primarily for diversity-oriented transformations by promiscuous glycosyltransferases.

37, 131-135, 198, 209

Free enzymes or whole

cell catalysts were used, typically in small scale reactions. Besides O-glycosides, a series of Nand S-glucosides were produced using para-nitrophenyl-β-D-glucoside as donor.

131-132, 135, 210


22

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ACS Catalysis

Rearrangement of aromatic O- to C-glycosides was also reported (Scheme 7B). This reaction is thermodynamically favored, thus enabling a highly atom-efficient transformation in quantitative 72, 142

yield.

5. INTEGRATED BIOCATALYTIC PRODUCTION OF GLYCOSIDES USING LELOIR GLYCOSYLTRANSFERASES Large-scale production (∼100 g) of glycosides by glycosyltransferases was interconnected with downstream processing in a holistic development of biocatalytic processes. Systematic evaluation of kinetic, thermodynamic and stability parameters of the enzymatic reaction(s) was essential to push the limits of the biocatalytic conversion. Thus, in the examples shown, process efficiencies typically required in fine chemicals production were obtained. 5.1 Nucleoside diphosphate-glucoses. A biocatalytic production of UDP-glucose was developed.

26, 211

Sucrose synthase converted sucrose and UDP into UDP-glucose and fructose

(Figure 2A). The logKeq for the reaction was shown to decrease at high pH above a pK of 6.0 (Figure 2B). Only when UDP was completely protonated at around pH 5.0, the UDP-glucose synthesis was thermodynamically favored. The Keq at pH 5.0 was 1.14. Under these conditions, with pH properly controlled during the reaction, UDP conversion was 85% when only a fivefold excess of sucrose was used. Recognizing the crucial role of pH in driving the reaction of sucrose synthase was essential to the identification of a narrow window of efficient process operation. 212

Sucrose synthases from bacteria (Acidithiobacillus caldus) 75

max)

26

and plant (soybean; Glycine

were compared for UDP-glucose production. In terms of kcat/Km, the plant sucrose 26

synthase was at least 45-fold more specific for UDP than the bacterial enzyme.

However, at

high UDP concentrations used in the reaction (300 mM), the specific activity of the A. caldus sucrose synthase exceeded that of the G. max enzyme by 11.4-fold. The effect was due to a combination of increased kcat and lowered substrate inhibition in the bacterial sucrose synthase. The A. caldus enzyme was therefore the clearly preferred choice for UDP-glucose production. -1

Gram-scale synthesis of UDP-glucose (255 mM; 144 g L ; Figure 2C-D) gave a productivity of


23

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-1

Page 24 of 45

-1

25 g L h . A mass-based TTN of 1440 was obtained for the sucrose synthase. These parameters were considered to be a good basis for further process development.

26

The optimized enzymatic reaction was integrated with efficient enzyme production and downstream processing.

211, 213

A. caldus sucrose synthase was expressed under control of a

constitutive promotor of intermediate strength. Bioreactor cultivation of E. coli BL21(DE3) gave -1

a total enzyme activity of 4800 U L culture. The sucrose synthase accumulated to a level of 480 U/g cell dry mass, which enabled direct use of mildly permeabilized E. coli cells as biocatalyst -1

for synthesis. The catalyst concentration used was only 2.5 g cell dry mass L . Synthesis at 100 -1

g scale was performed. The final UDP-glucose concentration was 100 g L and the yield based -1

-1

on UDP added to the reaction was 86%. The productivity was 10 g L h and the TTN based on dry cell mass used was about 100.

Figure 2. Enzymatic production of UDP-glucose by A. caldus sucrose synthase. A, Reaction scheme. B, Dependence of the equilibrium constant of the reaction (Keq) on pH at 45°C. C, Time -1

course of conversion using 1.5 M sucrose (5-fold excess over UDP) and 100 µg mL enzyme at 45°C and pH 5. The pH was controlled (with HCl) during the reaction. D, The substrates before dissolution in 100 mL total volume.


24

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ACS Catalysis

UDP-glucose was recovered from the reaction mixture in ≥90% purity and in 73% isolated yield. A chromatography-free procedure of downstream processing of nucleoside diphosphate glucoses was developed. This involved phosphatase digestion of residual UDP in the reaction mixture followed by ethanol precipitation of UDP-glucose as the main processing steps. The -1

overall balance for the biocatalytic process was about 0.7 kg UDP-glucose L E. coli bioreactor culture. Immobilization of sucrose synthase has only recently been accomplished and provides a significantly (340-fold) stabilized biocatalyst for UDP-glucose production that can be 77, 214

recycled.

The nucleoside diphosphate represents a main cost factor of glucose nucleotide production by sucrose synthase. Nucleoside monophosphate is significantly more expedient (≥ 100-fold in the case of AMP or UMP) as starting material. A biocatalytic cascade reaction was established for adenosine diphosphate glucose production from sucrose (1 M), adenosine 5'-monophosphate 118

(100 mM) and polyphosphate (132 mM).

Besides the A. caldus sucrose synthase, a

polyphosphate kinase (Scheme 5) from Meiothermus ruber 2+

performed at 45°C, pH 5.5 and 25 mM Mg

215

was applied. Optimized reaction -1

2+

gave ADP-glucose at 81 mM (48 g L ). The Mg

concentration was important not only for enzyme activity, especially that of polyphosphate kinase, but it also affected polyphosphate solubility. Using a total enzyme loading of just 150 mg -1

-1

-1

L , a productivity of 2 g L h was obtained and the mass-based TTN was 320. Purified sucrose synthase and polyphosphate kinase were employed in a 2:1 mass ratio. Isolation procedure for ADP-glucose in high purity (99%) was developed. Removal of polyphosphate was achieved using fractional precipitation with ethanol. ADP-glucose (155 mg) was purified in a (nonoptimized) yield of 30%. The biocatalytic cascade reaction by polyphosphate kinase and sucrose synthase could be applied to synthesize GDP-glucose, CDP-glucose, UDP-glucose and dTDPglucose from the corresponding nucleoside monophosphates (10 mM). Its efficiency decreased in the order of glucose nucleotides shown, reflecting preference of the polyphosphate kinase for reaction with pyrimidine as compared to purine nucleobases.


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Page 26 of 45

A potentially interesting catalyst for glucose nucleotide synthesis is trehalose synthase. Using the enzyme from the hyperthermophile Pyrocococcus furiosus at 37°C and applying nucleoside diphosphate (113 - 117 mM) and trehalose (420 - 444 mM) in excess, production of ADP-Glc (35%; 41 mM), GDP-Glc (17%; 19 mM), CDP-Glc (12%; 14 mM) and UDP-Glc (10%; 11 mM) -

was shown in repeated cycles (up to 30; 12 h each) with re-use of the soluble enzyme (0.24 g L 1

). A mass-based TTN of well above thousand appears to be possible with this apparently quite 216

stable enzyme.

5.2 Nothofagin. The natural dihydrochalcone phloretin occurs in different glycoforms, typically featuring β-D-glucosylation of one phenolic hydroxy group. In addition, C-glucosylation at the C-3' of phloretin is found in nothofagin, a natural product present in the rooibos plant and linked 217

to the various health-promoting effects of its herbal tea.

Nothofagin represents an emerging

class of C-glycosidic natural products, which have recently attracted much interest for their 38, 218-220

different possible applications.

Nothofagin is a powerful antioxidant and has positive

effects in diabetes treatment. Natural scarcity and difficult chemical synthesis restrict promising applications of nothofagin. Biocatalytic synthesis was therefore considered. Installing the Cglycosidic linkage in nothofagin requires Leloir glycosyltransferases distinct from the enzymes 221

synthesizing phloretin O-glucosides. Using the C-glucosyltransferase from rice (Oryza sativa)

a biocatalytic synthesis of nothofagin at 100 g scale was developed. Promising design for the enzymatic reaction involved a cascade of the C-glucosyltransferase and sucrose synthase, as 75

shown in Figure 3A. The one-pot β-D-glucosylation at C3' of phloretin occurs from sucrose via an UDP/UDP-glucose shuttle. The same concept was used for synthesis of phloretin-Oglucosides.

222

Kinetic characterization of different sucrose synthases revealed that a low UDP Km was very 86

important for enzyme efficiency in UDP-glucose regeneration.

The G. max sucrose synthase

was superior to the A. caldus sucrose synthase according to this kinetic criterion. The A. caldus enzyme preferred ADP over UDP. Structure-guided engineering of the sucrose synthase was 223

used to enhance the enzyme's affinity for UDP.

Engineered variants of the A. caldus enzyme

exhibiting a UDP Km 60-fold lowered compared to the wildtype sucrose synthase showed


26

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ACS Catalysis

86

improved performance in UDP-glucose recycling.

However, the G. max sucrose synthase,

which naturally exhibits a high affinity for UDP (Km = 0.13 mM) was the best enzyme candidate for coupling with the C-glycosyltransferase. Basic parameters of the cascade reaction requiring optimization were the enzyme activity ratio (5-fold excess of C-glycosyltransferase) as well as the sucrose (100 mM) and the UDP concentration (0.5 - 1.0 mM). The low water-solubility of phloretin (≤ 0.5 mM) was a main bottleneck of an efficient nothofagin synthesis. Using DMSO (20%, by volume) as cosolvent, about 5 mM phloretin was dissolved and the same concentration of nothofagin was produced (Figure 3B). To enhance the final nothofagin concentration, phloretin was added in 5 mM portions during the course of the 75

reaction and about 45 mM product could so be formed (Figure 3B). Among various strategies for phloretin solubility enhancement, inclusion complexation by β-cyclodextrins was best compatible with enzyme stability and activity (Figure 3B).

79

The expedient and well-soluble 2-

hydroxypropyl-β-cyclodextrin enabled solubilization of phloretin to a point (∼150 mM) where 76

viscous fluid mixing became a physical boundary on the process operation.

The nothofagin

synthesis thus pushed to upper practical limits gave a final product concentration of ∼120 mM -1

-1

-1

(50 g L ) at a productivity of about 3 g L h (Figure 3B-C). The UDP was regenerated 220 times. A limiting mass-based TTN of 100 was obtained for the C-glycosyltransferase in a single batch conversion lasting 24 h (Figure 3C). Reaction scale up to the 50 - 100 g scale was done with no practical loss in efficiency and productivity (Figure 3D). Stability of the enzymes, especially the sucrose synthase, remains an issue. Immobilization of the sucrose synthase, or of both enzymes, could present a useful solution for stabilization and re-use.

77, 214

Isolation of polyphenol glycosides from glycosyltransferase reactions was realized previously mostly at small preparative scale (≤ 100 mg). It involved procedures, like size exclusion chromatography, that are not easy to scale up. A downstream process for efficient recovery of 76

nothofagin (≥ 95% purity) was developed.

A tailored anion-exchange chromatography at pH

8.5 was used for capture and initial purification of the product. The 2-hydroxypropyl-βcyclodextrin could be also recovered at this step. Precipitation of nothofagin at a lowered pH of 6.0 and re-dissolution in acetone effectively replaced desalting by size exclusion chromatography


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76

in the final step of product purification. Nothofagin was recovered in an overall yield of 65%.

The successful nothofagin process involves upstream and downstream processing efficiently interconnected. The idea of a holistic process development for biocatalytic production of flavonoid glycosides by coupled Leloir glycosyltransferases is supported. This may find further applications in natural product glycosylation. By including additional enzymatic steps into the reaction cascade (e.g., epimerisation, oxidation at C'6), the scope of glycosylation from sucrose can be expanded to glycosyl residues (e.g., Gal or GlcA) other than Glc.

A

B

C

D

61

Figure 3. Nothofagin synthesis from phloretin and sucrose by C-glycosyltransferase (from O. sativa; OsCGT) and sucrose synthase (from G. max; GmSuSy) at pH 6.5 and 40°C. A, Reaction scheme. B, Optimization of the nothofagin titer (blue) from initially 5 mM using (left to right) substrate feeding and inclusion complexation with β-cyclodextrin and 2-hydroxypropyl-βcyclodextrin. Product yield is shown in black. C, Time course of nothofagin production at 50 g -1

-1

scale using 1.6 units mL C-glycosyltransferase and 0.5 units mL sucrose synthase. Reaction


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conditions: 112 mM phloretin (complexed with 140 mM 2-hydroxypropyl-β-cyclodextrin), 500 mM sucrose, 0.5 mM UDP. D, Scale up effect on space-time yield and product yield.

6. CONCLUSIONS In comparing known catalysts for glycosylation, Leloir glycosyltransferases impress with their selectivity. In addition, many glycosyltransferases exhibit remarkably flexible substrate specificity, rendering them broadly applicable for synthesis. To harness the prowess of glycosyltransferases for chemical production, limitations in robustness and efficiency of their corresponding biocatalytic systems need to be overcome. Enhancing enzyme expression to enable efficient whole-cell biocatalysis is key. A comprehensive approach integrating enzyme engineering with synthetic biology will be required. Biochemical engineering principles applied to the characterization of glycosyltransferase cascades will facilitate optimization of their output 224-225

in one-pot multicomponent transformations. Adopting concepts of flow chemistry

might

enable continuous production with integrated enzyme re-use. A holistic process design involves a biocatalytic synthesis optimized for efficient interconnected performance with product recovery. Perceived limitations on the applicability of Leloir glycosyltransferases in industrial biocatalysis will be challenged most effectively through paradigmatic examples of their successful use.

ASSOCIATED CONTENT Supporting Information. Tables S1 and S2 giving a representative summary of recombinant production

in

E.

coli

of,

respectively,

bacterial

sialyltransferases

and

bacterial

fucosyltransferases. This information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author


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*Bernd Nidetzky; Institute of Biotechnology and Biochemical Engineering, TU Graz; Petersgasse 12, A-8010 Graz, Austria; e-mail: [email protected]; phone: +433168738400; fax: +433168738434 Present Addresses Alexander Gutmann, Specialty Ingredients & Technology, Lonza AG, Lonzastraße, CH-3930 Visp, Switzerland Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources EU FP7 project SuSy (Sucrose Synthase as Effective Mediator of Glycosylation); Austrian Research Promotion Agency FFG through the COMET Funding Program.

ACKNOWLEDGMENT Funding from the EU FP7 project SuSy (Sucrose Synthase as Effective Mediator of Glycosylation) and the Austrian Research Promotion Agency FFG through the COMET Funding Program is gratefully acknowledged.

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ACS Catalysis

GRAPHICAL ABSTRACT

X = O, N, S, C

HO

O NDP

H + X

= natural product, sugar, oligosaccharide

HO

O X

+

NDP-O(H)

O Recycle cascade

Integrated biocatalytic glycosylation process for chemical production

The development of Leloir glycosyltransferases into optimized biocatalytic systems for practical use in synthetic glycosylations is reviewed. Strategies to overcome perceived limitations on the applicability of Leloir glycosyltransferases are illustrated from first examples of integrated biocatalytic process development with this class of enzymes.


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Leloir Glycosyltransferases as Biocatalysts for ... - ACS Publications

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