D -Serine as a Key Building Block: Enzymatic Process Development and Smart Applications within the Cascade Enzymatic Concept

Article abstract of DOI:10.1021/acs.oprd.0c00024

An efficient enzymatic method catalyzed by an enzyme from the d-threonine aldolase (DTA) family was developed for d-serine production at industrial scale. This process was used for the synthesis of two valuable ketoses, l-erythrulose and d-fructose, within the cascade enzymatic concept involving two other enzymes. Indeed, d-serine was used as a substrate of d-amino acid oxidase (DAAO) for the in situ generation of the corresponding α-keto acid, hydroxypyruvic acid (HPA), a key donor substrate of transketolase (TK). This enzyme catalyzed the irreversible transfer of the ketol group from HPA to an aldehyde acceptor to form a (3S)-ketose by stereoselective carbon-carbon bond formation. The compatibility of all enzymes and substrates allowed a sequential three-step enzymatic process to be performed without purification of the intermediates. This strategy was validated with two TK aldehyde substrates to finally obtain the corresponding (3S)-ketoses with high control of the stereoselectivity and excellent aldehyde conversion rates.

Full text of DOI:10.1021/acs.oprd.0c00024

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Full Paper
D-Serine as a Key Building-Block : Enzymatic Process Development
&
Smart Applications within Cascade Enzymatic Concept
Nazim Ocal, Mélanie L'enfant, Franck Charmantray, Loredano Pollegioni,
Juliette MARTIN, Pascal Auffray, Jérôme Collin, and Laurence Hecquet
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.0c00024 • Publication Date (Web): 04 May 2020
Downloaded from pubs.acs.org on May 4, 2020
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D-Serine as a Key Building-Block : Enzymatic
Process Development & Smart Applications within
Cascade Enzymatic Concept
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Nazim Ocal,† Mélanie L’enfant,† Franck Charmantray,† Loredano Pollegioni,§
Juliette Martin‡*, Pascal Auffray‡, Jérôme Collin‡ Laurence Hecquet†*
†
Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de
Clermont-Ferrand (ICCF) F-63000 Clermont-Ferrand (France)
‡
§
Protéus by Seqens, 70 Allée Graham Belln F-30035 Nîmes (France)
Department of Biotechnology and Life Sciences, Università degli Studi dell’Insubria,
Varese (Italy)
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TOC graphic
step 1
DTA
DTA
NH2
O
+
DTA
COOH
H
H
E.Coli cells
overproducing DTA
Glycine Formaldehyde
step 2
step 3
TK
DTA
DAAO
O
O
NH2
HO
HO
R
HO
COOH
COOH
H2O2 +NH4+
OH
(3S)-Ketose
1
/2 O2
O
CO2
HPA
R
D-Serine
Catalase
Simultaneous 2 and 3 steps
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ABSTRACT. An efficient enzymatic way catalyzed by an enzyme from D-Threonine Aldolase
family was developed for D-serine production at industrial scale. This process was used to the
synthesis of two valuable ketoses, L-erythrulose and D-fructose, within cascade
enzymatic concept involving both other enzymes. Indeed, D-serine was used as a substrate
of D-amino acid oxidase for the in situ generation of the corresponding ketoacid,
hydroxypyruvate, a key donor substrate of Transketolase. This enzyme catalyzed the
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irreversible transfer of the ketol group from hydroxypyruvate to an aldehyde acceptor to
form a (3S)-ketose by stereoselective carbon-carbon bond formation. The compatibility
of all enzymes and substrates allowed to perform a sequential three step enzymatic
process avoiding the purification of intermediates. This strategy was validated with two
TK aldehyde substrates to finally obtain the corresponding (3S)-ketoses with a high
control of the stereoselectivity and excellent aldehyde conversion rates.
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KEYWORDS. D-Serine, D-Threonine Aldolase, D-Amino Acid Oxidase, Transketolase,
Ketoses
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INTRODUCTION
Small chiral molecules in the pharmaceutical pipeline becoming more and more complex,
their asymmetric synthesis requires multistep processes. The mild conditions and the high
specificity of enzymes have led to the development of efficient biocatalytic strategies in
the synthesis of bulk chemicals and drugs.1,2
Enzymatic one pot synthesis provide competitive strategies to the chemical approaches,
by using simultaneously two or more enzymes in cascade avoiding the isolation of
intermediates, thus saving time, energy, and limiting waste. The drawbacks of this
procedure such as inhibition of the substrate / product / reagent or the incompatibility of
the reaction condition, can be circumvented by a sequential one-pot procedure.3
For natural and unnatural aminoacids and monosaccharides synthesis, enzymatic
reactions are particularly useful to generate asymmetric centers with high
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stereoselectivity, avoiding protection and deprotection steps required by the chemical
ways. Enzymes catalyzing a stereospecific carbon-carbon bond formation such as
aldolases and transketolases became very useful for obtaining such compounds.4
Amoung these enzymes, threonine aldolases (TA) have been particularly used for the synthesis
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of optically pure natural and non-natural amino acids. In the alternative pyridoxal-5′-
phosphate (PLP) synthesis pathway, TAs catalyze the retro-aldol cleavage of threonine to form
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glycine and acetaldehyde. L- or D-TAs are highly selective at the α-carbon of threonine but, only
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a few native DTA genes have been described. The catalytic mechanism of these enzymes needs
2
+
2+
2+
2+
PLP and divalent metal ions such as Co , Ni , Mn or Mg as a cofactors. For biocatalytic
applications, the reversibility of the retro-aldol TAs-catalyzed reaction is of great interest for the
synthesis of optically pure natural and non-natural amino acids. The synthetic reactions and the
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substrate scope of TAs from different microorganisms were recently reported. These enzymes
have notably been used for the synthesis of L- and D-β-hydroxy-α-amino acids starting from
achiral aldehydes and glycine in creating two chiral centers.9
In the area of monosaccharides synthesis, beside enzymatic aldose-ketose
isomerization,10 transketolases (TK EC 2.2.1.1) are also popular.11 TKs are thiamine
diphosphate-dependent enzymes, and catalyze the transfer of a two-carbon unit (ketol
group), from a donor to an aldehyde acceptor, leading to a Cn+2 (3S)-ketose by the
stereoselective formation of the C2-C3 bond. (Scheme 1) In pentose phosphate metabolic
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pathway, TK reversibly transfers a ketol group from a ketose phosphate to an aldose
phosphate. Previous in vitro studies showed that TK accepts non-phosphorylated
substrates leading to the corresponding free hydroxyketones. Particularly,
hydroxypyruvate (HPA) can be used as donor rendering the reaction irreversible due to
carbon dioxide release. This major advantage has been largely applied in the presence
of various aldehydes as acceptors for the synthesis of corresponding ketoses in one step.
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(
Scheme 1).11
NH2
CO H

-ketoacid
A
HO
2
-aminoacid
L-serine
TA
TK
O
O
HO
HO
+
CO2
CO H
R
2
DAAO
OH
Product
NH2
Donor
HPA
R
H O
B
HO
2
2
O
CO H
+ NH4+
2
O2
(3S)-hydroxyketose
Acceptor
aldehyde
D-serine
Scheme 1: Cascade reactions involving Transaminase or D-Amino Acid Oxydase-
catalyzed reactions coupled with TK for in situ generation of HPA.
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The main problems of the TK-catalyzed reaction for biocatalytic applications at industrial-
scale, are in one hand the HPA synthesis, commonly obtained from toxic bromopyruvic
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acid in the presence lithium hydroxide, with a moderate yield and in the other hand, its
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unstability in water at temperature superior at 25°C. To circumvent all these limitations,
enzymatic in situ generation of HPA has been previously reported, in particular from L-
serine using transaminases 14 and more recently, from D-serine using D-amino acid
oxidase (DAAO) (Scheme 1).15 This last way offers major advantages compared to
transaminase strategy, such as an irreversible reaction and no requirement of an
additional substrate.
step 1
E3005
step 2
DAAO
step 3
TK
NH2
COOH
O
O
NH2
O
+
HO
D-Serine
HO
HO
R
COOH
COOH
Glycine
H
H
+
OH
3S)-Ketose
1/2 O2
H O +NH4
2
2
O
CO2
HPA
R
Formaldehyde
(
Catalase
Sequential step 1
Simultaneous 2 and 3 steps
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Scheme 2: One pot three-steps enzymatic cascade catalyzed by a crude extract of E. coli
strain P3005 and purified D-amino acid oxidase from Rhodotorula gracilis (DAAO ) and
Rg
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transketolase from Geobacillus stearothermophilus (TK ) with three different TK
gst
aldehyde substrates : glycolaldehyde (R = -CH OH), and D-erythrose (R= -CHOH(R)-
2
CHOH(R)-CH OH).
2
In this work, we first carried out a detailed investigation of D-serine production at industrial
scale catalyzed by DTA overexpressed in Escherichia coli strain from achiral compounds
glycine and formaldehyde. This strategy was then applied in a sequential three step
cascade enzymatic concept for the synthesis of two valuable (3S)-ketoses (Scheme 2).
In a first step, D-TA cell free extract produced D-serine. Then, two simultaneous
enzymatic steps were developed at laboratory scale. DAAO from Rhodotorula gracilis
16
(DAAO ) transformed D-serine into the corresponding ketoacid, hydroxypyruvate used
Rg
13
by TK from Geobacillus stearothermophilus (TK ) in the presence of an aldehyde as
gst
acceptor for obtaining the ketose product. The one pot three steps process was validated
using three TK aldehyde acceptors, glycolaldehyde and D-erythrose for the synthesis of
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corresponding valuable (3S)-ketoses, L-erythrulose, a tanning agent in the cosmetics
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industry, and D-fructose, a sweetener with very low glycemic index.
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RESULTS AND DISCUSSION
D-serine Enzymatic Process Development from whole cells
The first step of the cascade reaction consists in the production of D-serine from glycine
and formaldehyde as substrates with DTA (P3005) enzyme overproduced in Escherichia
coli strain (whole cells). This step was developed by Seqens Company and applied to the
production of D-serine in an enantiomerically pure form at multiton scale per year. The
enzyme candidate DTA (P3005) was selected among off-the-shelf enzymes portfolio of
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Seqens company which provided high level of enantioselectivity (ee >99%) required for
fine chemicals application.
NH2
D-TA E3005 (in E.coli)
HOOC
HO
NH2
COOH
HCHO (37% aq.)
PLP, MgCl2
30°C
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Scheme 3: Enzymatic aldolization of glycine with formaldehyde by biocatalyst DTA P3005
expressed in E. coli for selectively producing D-serine.
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Firstly, the fermentation process of Escherichia coli overproducing DTA was developped
to ensure production in large fermenters (Figure 1 and SI). The optimal conditions led to
55 g of wet cells per liter of culture medium. The figure below is a representation of
successive steps required for the large manufacturing fermentation process.
Main
Fermentation
Intermediate
Fermentation
WCB
Seeding
0.1%
1%
Centrifugation
2%
E.coli cells
over producing
DTA
1
mL
200 mL
10 L
10% dry
matter
Cryovial
1 mL
1
L in 5-L flask
6 h
1
1
0 L in 20-L vessel
8-16 h
3
3
6
m medium + 3m
3
feed in 15m vessel
40-50 h
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Figure 1: Different steps of fermentation process flow for the manufacturing production of
E.coli cells.
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At first, we developed the fermentation process of the DTA enzyme overexpressed in
Escherichia coli. To define the fermentation process, a Statistical Experimental Design
has been carried out in order to determine the critical and important parameters, such as
oxygen concentration, glucose feed and its optimal concentration, and other medium
components impact. This approach allowed us to determine a robust domain of
operations for producing the overexpressed enzyme. The Fermentation at different
scales, from lab scale (1 liter) to several cubic meters volume fermenter size (up to
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working capacity of 9 m ), have been implemented successively before ensuring
production in large fermenters (Figure 1).
The biomass (whole cells) was then used for the synthesis of D-serine from glycine and
formaldehyde. The optimal conditions to ensure high performance, high productivity and
robustness for multi-ton manufacturing of D-serine were determined (Table 1).
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Process parameters
Optimal values
7 ± 0,5
pH
MgCl2
41.2 mM
2.12 mM
1.33 M
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
4
4
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4
4
4
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
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5
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7
8
9
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1
2
3
4
5
6
7
8
9
0
PLP
Glycine
Formaldehyde
Base
1,2 M
KOH (0.3 M)
30°C
Temperature
Table 1: Optimal conditions for glycine and formaldehyde biotransformation into D-serine
by whole cells overproducing DTA.
-1
The reaction was carried out in water with glycine (1.33 M, 300g.L ) and formaldehyde
-1
(
37% aq. 1.2 M) with E. coli P3005 cells (0.6 g of wet cells.L ).
PLP was the cofactor required for the catalytic mechanism of DTA and a better conversion
rate was observed when 2.12 mM of PLP was added in the medium (SI, chart 1). In
addition, the optimal temperature was 30°C because at higher temperature (40-50°C),
the conversion rate was rapidly limited by the degradation of formaldehyde and the
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1
1
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2
2
2
2
2
2
2
2
2
3
3
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formation of significant amounts of by-products (data not shown) yielding to an inhibition
of the enzymatic activity (SI, Chart 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The effect of pH in the 6-8 range has also been investigated showing that the optimal conversion
of substrates was obtained at pH 7 - 7.5 (SI, Chart 3). The pH must be continuously controlled
during the reaction because an acidification occurs probably due to the formaldehyde addition.
Indeed, typical commercial aqueous solution of formaldehyde 37% has a pH of 3 (certainly due to
residual formic acid presence). Finally, in order to avoid local racemization during the feed of the
base solution, we determined the best compromise between base nature impact on the reaction rate
and the enantiomeric excess of D-serine product. The influence of three different bases was studied
(
SI, Chart 4). NH OH and KOH solutions gave the best conversion rates avoiding potential
4
racemization of D-serine. The conversion in the presence of KOH was significantly better and did
not exhibit any side by-products, according to the HPLC analysis.
Using these optimal conditions (Table 1), D-serine was produced at kg scale. After 24 h
both substrates were almost fully converted (90 %). The reaction mixture was then
concentrated by heating to 40°C under vacuum followed by the elimination of the whole
cells by centrifugation. The crude residue containing D-serine was treated with charcoal
to remove the salts and then filtered. Finally, a recrystallization in a water/alcohol mixture
gave pure D-serine with an isolated yield of 70 %. with an excellent enantiomeric excess
of 99% determined by HPLC. This process offers many advantages compared to the other
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microbial procedures21 such as full conversion of achiral, cheap and affordable
substrates, no requirement of enzyme purification, and high stereoselectivity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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0
1
2
3
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8
9
0
1
2
3
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8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Smart Application to ketose production within cascade enzymatic concept
For the cascade enzymatic concept, the first step was the production of D-serine from
glycoladehyde and glycine and was conducted with DTA cell free extract from E.coli cells
obtained previously. This reaction was performed independently because formaldehyde
is an inhibitor of DAAO used in the second step and can also be a substrate of TK used
in the third step. The DTA (cell free extract)-catalyzed reaction was studied using 50 mM
and 100 mM substrate concentrations (Table 2). Lower concentrations were not chosen
because we previously showed that the best result for the D-serine conversion catalyzed
by DAAORg was obtained at 50 mM.15
Table 2. Influence of substrate concentrations on D-serine production using DTA (cell free
extract,CFE)a.
Substrates (mM)
Reaction
Glycine
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1
2
2
2
2
2
2
2
2
2
2
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3
3
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Glycine
Formaldehyde
time (h)°
24h
conversion rate (%)b
50
50
94
82
0
1
2
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8
9
0
1
2
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9
0
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0
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7
8
9
0
100
100
24h
a
b
1
DTA CFE (3.5 mg of proteins) ; determined by in situ HNMR
A concentration of 50 mM for both substrates was used to perform the reaction because
an almost complete substrate conversion of both substrates (94%) was obtained after
2
4h against 82% with 100 mM of both substrates. The reaction mixture was treated with
methanol to precipitate proteins and to avoid by-products formation from substrates and
reagents used in steps 2 and 3. After filtration and methanol evaporation, D-serine was
used in the second part of the process in which both DAAORg and TKgst enzymes were
coupled for the synthesis of the ketoses.
DAAORg,16 overexpressed in E. coli and purified by Ni2+ chelating affinity column
chromatography as reported earlier (SI), was used to convert D-serine into HPA. The
choice of this enzyme can be explained by its broad amino acid spectrum including D-
22
serine while the best substrates for L-AAOs are hydrophobic amino acids. In addition,
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DAAORg showed much higher turnover number, better stability under different reaction
conditions, and a larger active site to accommodate various substrates than the other
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
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0
1
2
3
4
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8
9
0
1
2
3
4
5
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8
9
0
1
2
3
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0
1
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3
4
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6
7
8
9
0
23
source of DAAOs. DAAOs (EC 1.4.3.3) are flavin adenine dinucleotide (FAD)-containing
flavoenzymes that catalyze the deamination of D-amino acids to their corresponding
imino acids. FAD is concomitantly reducted and then re-oxidized by molecular oxygen
with generation of hydrogen peroxide. The released imino acid spontaneously led to the
corresponding -keto acid and ammonia (Scheme 2).
The thermostable TKgst was selected for its properties compatible with industrial
applications such as high stability and robustness against unusual conditions, reusability
24
25
after immobilization and evolvability. TK was overexpressed in E. coli and purified
gst
2+
13
by Ni chelating affinity column chromatography as reported earlier (SI).
The strategy was based on the HPA in situ generation by DAAO from -serine (obtained
D
Rg
previously with DTA) and its use as donor substrate by TK catalyzed reaction in the
gst-
presence of an aldehyde as acceptor. To validate this simultaneous two-step cascade
reaction, two aldehydes having an increased carbon chain length were chosen, such as
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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glycolaldehyde C (1), and D-erythrose C (2) leading to the corresponding valuable C
2
4
n+2
ketoses, L-erythrulose C (3), and D-fructose C (4) respectively (Table 3).
4
6
0
1
2
3
4
5
6
7
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9
0
1
2
3
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9
0
1
2
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9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
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9
0
The influence of enzyme quantity and TK aldehyde substrate concentration were studied
and the best conditions are indicated in table 3.
Table 3. Results obtained by coupling DAAORg and TKgst for ketoses 3 and 4 synthesis
from aldehydes 1 and 2 and D-serine.
TK
TK products
Ketoses
DAAO /TK
(mg)a
Aldehyde Reactio Isolated
e.e. or
d.e.
Rg
gst
acceptors
Aldehydesa
conversio
n
product
n
Time
yield (%) (%)d
rate (%)b
(h) c
O
OH
O
HO
OH
2
/6
/6
82
8
8
60%
57%
>95%
OH
3
1
OH
O
OH
2
>95
>95%
HO
OH
OH
O
OH
OH OH
2
4
a
Reactions were carried out with DAAORg (2 mg, 86 U) and TKgst (6 mg, 42 U), ThDP
b
(
0.1 mM), MgCl (1 mM), aldose (50 mM), D-serine (50 mM) at pH 7 and 25°C. Alehyde
2
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acceptor conversion determined by in situ H NMR analysis. c 1H NMR of reaction mixture
I
d
1
(
SI). Enantiomeric excess (e.e.), diastereoisomeric excess (d.e.) determined by H NMR.
According our previous studies, D-serine and aldehyde 1 or 2 were used at a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
concentration of 50 mM. A minimum amount of DAAO and TK was used and the ratio
Rg
gst
of both enzymes allowed a total conversion of D-serine avoiding the accumulation of HPA.
Hence, the bi-enzymatic cascade led to an almost total (82 % and 95 % for 1 and 2
1
respectively) and rapid (8 h) conversion of both substrates as observed by in situ H NMR
(
Table 2). The purification and the characterization of ketoses 1 and 2 showed high
enantio- and diastereoselectivity (> 95 %) and good isolated yields (57-60 %).
CONCLUSIONS
An efficient enzymatic way catalyzed by D-TA overproduced in Escherichia coli was first
developed in whole cells for D-serine production at industrial scale offering many advantages
21
compared to the other microbial procedures such as full conversion of achiral, cheap and
affordable substrates, no requirement of enzyme purification, and high stereoselectivity. Then, a
smart application of DTA-catalyzed process using crude extract was applied to ketose
production within cascade enzymatic concept involving both other enzymes, DAAO and
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
6
TK. The three enzymatic steps were performed with cell free extracts for the synthesis of
two valuable (3S)-ketoses, L-erythrulose 3 and D-fructose 4 with good yields and
excellent stereoselectivities. This process allowed to resolve the major drawback of TK-
catalyzed reaction for potential industrial applications. Indeed, HPA the key TK donor
substrate rendering the reaction irreversible was in situ generated by DTA and then
DAAO from achiral glycine and formaldehyde according anenvironmentally friendly
procedure. This strategy offers a competitive way to the commonly used chemical
synthesis of HPA from bromopyruvate12 and to the in situ HPA generation by
transaminase-catalyzed reaction.14c,d Since the three enzymes are active under similar
conditions, this cascade synthesis avoided the isolation of intermediates and reduced
waste.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The entire process could be further optimized, especially by co-immobilization of all enzymes to
ensure their reusability and by performing the conversion in a continuous reactor for higher scale
production of ketoses. The procedure could also be extended to TK variants or to other HPA-
dependent enzymes in place of wild-type TK for obtaining a wide range of (3S)-ketoses and
analogs.
EXPERIMENTAL SECTION
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General. All chemicals were purchased from Sigma-Aldrich, Alfa-Aesar and CarboSynth.
Bradford reagent was from Bio-Rad. Ni-NTA resin was obtained from QIAGEN. Lyophilization
was carried out with Triad LABCONCO dryer. UV-visible absorbance was measured using a
Spark control 10 microplate reader from TECAN and an Agilent Technologies, Cary 300 UV-Vis
spectrophotometer enabling Peltier temperature control. Liquid Flash Chromatography were
performed with MARCHEREY-NAGEL GmbH & Co KG 60/40-63 mesh silica gel and for Thin
Layer Chromatography MARCHEREY-NAGEL GmbH & Co KG 60 F254 silica gel TLC plates
with anisaldehyde stain for detection was used. A pH autotitrator (TitroLine 7000) allowed to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
continuously adjust the pH of the reaction. NMR spectra were recorded in D O or DMSO on a
2
4
00MHz Bruker Avance III HD spectrometer. Chemical shifts are referenced to the residual
solvent peak. The following multiplicity abbreviations were used: (s) singlet, (d) doublet, (t) triplet,
m) multiplet. DTA-catalyzed reaction was monitored with HPLC Agilent with a Spherisorb
(
-1
column (CN – 250 x 0.46 – 5 m – 80 Å) and a flow at 0.2 mL . min (eluent: aqueous formic
acid 0.05%). The enantiomeric excess of serine produced with DTA from glycine and
formaldehyde was determined with HPLC (waters 2487) with a Crownpack column (Cr(-), 5 μm
-1
,
150 x 4 mm), using a flow at 0.2 mL . min (MeOH/HClO aq (pH 1) : 10/90), at 30°C, and a
4
detection at 200 nm.
1
In situ H NMR measurements. Progress of preparative scale enzymatic synthesis were
monitored by using quantitative in situ 1HNMR relative to 3-trimethylsilyl-2,2,3,3-
tetradeuteropropionate (TSP-d ) as internal standard. Aliquots of the reaction mixture were
4
removed overtime (450 µL) and mixed with 50 µL of TSP-d (50 mM in D O).
4
2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Obtention of E.coli cells overproducing DTA. Escherichia coli strain BL21DE3 was used for
DTA overexpression with the plasmid pET26b+. This strain was incubated at 37°C, 180 rpm for
16h in LB medium containing kanamycin at 30 µg/mL (PC1). 0.2 liter of the pre-culture was used
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
to inoculate 1 liter of culture medium containing 30 µg . mL of kanamycin and grown at 37°C,
1
80 rpm. Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 0.15 mM was added when the OD600nm
was 0.7-0.8. The cells were then grown overnight at 37°C, under stirring at 180 rpm, and harvested
by centrifugation at 3600 g at 4°C for 30 min. Bacterial pellets were frozen and stored at -80°C
-1
(
200 g of wet cells . L ).
D-serine production from whole cells at industrial scale. Glycine (4.4 M, 13.3 mol, 1 kg),
formaldehyde (4 M, 12 mol, 0.9 kg), PLP (6.7 mM, 0.02 mol, 5 g), MgCl (13.3 mM, 0.04 mol,
2
-1
0
.08 kg) were introduced in a suspension of E.coli cells (0.02 kg cells . L ) at 30°C. The pH was
continuously adjusted at 7-7.5 with KOH (1 M). After 24 hours, the reaction mixture was then
acidified at pH 6 with a solution of phosphoric acid (0.7 M) and then concentrated (at 40°C
max) by reaching a final volume of 4 liters. The whole cells were then eliminated by
filtration on charcoal adjuvant. The crude residue containing D-serine was treated with
charcoal to remove the salts and then filtered. Finally, a recrystallization in a water/alcohol
mixture gave pure D-serine as a white powder with an isolated yield of 70 % and with an
excellent enantiomeric excess of 99% determined by HPLC.
Obtention of cell free extract from E.coli whole cells. Harvested recombinant cells from 1 liter
of culture were resuspended in 50 mL of phosphate buffer (0.2 M) at pH 7. The cells were disrupted
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with a French Press at 2 kbar. After sonication of the lysate (3 cycles of 30 seconds each at 60W
on ice), cellular debris were removed by centrifugation during 30 min at 20 000 g. The supernatant
was centrifuged a second time in the same conditions.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D-serine synthesis catalyzed by cell free extract D-TA P3005. To the solution containing
glycine and formaldehyde (50 mM, 1 mmol, 30 mg) or (100 mM, 2 mmol, 60 mg) were added
PLP (0.05 mM, 0.001 mmol, 0.25 mg), MgCl (10 mM, 0.2 mmol, 19 mg) and cell free extract of
2
D-TA P3005 (133µL of CFE containing 3.5 mg of proteins) in H O. The pH was adjusted at 7.3
2
with 0.1M NaOH. After 24 h, methanol (twice the reaction mixture volume) was added. After 16
h at 4°C, the solution was filtrated and evaporated under reduced pressure. D-serine was
1
characterized by HPLC and H NMR (SI) and was obtained as a white solid with 97% yield.
Synthesis of ketoses 3 and 4 catalyzed by purified TK and DAAO . ThDP (0.1 mM, 0.002
gst
Rg
mmol, 0.85 mg), MgCl (1 mM, 0.02 mmol, 1.9 mg), D-serine previously produced using DTA
2
(50 mM, 1 mmol, 105 mg) and glycolaldehyde (50 mM, 1 mmol, 60 mg) or D-erythrose (50 mM,
1
mmol, 120 mg) were dissolved in H O and the pH was adjusted to 7 with 0.1 M NaOH. In this
2
reaction mixture were introduced catalase (1 mg / 2 h, 1998-4995 U / 2 h), DAAO (2 mg, 86 U).
Rg
TK was then added (6 mg, 42 U) in three portions (2 mg, 14 U at initial time, 2mg, 14 U at 2 h
gst
3
0 and finally 2 mg, 14 U at 5 h) giving a final volume of 20 mL. Oxygen was bubbled into the
.
-1
reactor (10 mL min at atmospheric pressure). The reaction mixture was stirred (100 rpm) at
2
5°C and the pH was automatically maintained at 7 by adding 0.1 M HCl using a pH stat. D-serine
1
and aldehydes consumption were monitored by in situ H NMR and TLC. The reactions were
stopped after total conversion aldehyde and enzymes were removed by precipitation and
centrifugation. Silica was added to the supernatant and the suspension was concentrated to dryness
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under reduced pressure. The powder was loaded onto a flash silica column. Products 3 and 4 were
purified using eluent CH -COO(C H )/CH OH (9/1) and CH Cl /CH OH (9:1) respectively.
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(3S)-1, 3, 4-trihydroxy-butan-2-one 2 (L-erythrulose) 3. Compound 3 was isolated as a yellow
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oil; yield : 160 mg (44%). []D25= +6.3 (c 1.48, H O), lit. [] = +6.3 (c 1.1, H O), []D25= +6.7
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2
D
2
(c 1.48, H O). NMR spectra were identical to those previously described.28 1H NMR (400 MHz,
29
2
MeOD): d=4.52 (d, J=19.3 Hz, 1H, 1-HA), 4.45 (d, J=19.3 Hz, 1H, 1-HB), 4.26 (t, J=4.2 Hz, 1H,
1
3
3
-H), 3.80 (dd, J=11.6, 4.5 Hz, 1H, 4-H), 3.76 (dd, J=11.6, 4.0 Hz, 1H, 4-H); C NMR (101 MHz,
MeOD): d=212.98 (C-2), 77.82 (C-3), 67.75 (C-1), 64.86 (C-4). m/z HRMS found [M+HCOO]-
165.0405, C H O requires 165.0396.
5
9
6
(3S,4R,5R)-1,3,4,5,6-pentahydroxyhexan-2-one (D-fructose) 4. Compound 4 was isolated as
colorless oil (118 mg, 65% yield). NMR data were identical to those previously described
(ratio :73/22/5; lit. Ratio : 75/21/4).30,31 1H NMR (400 MHz, D O) : δ (ppm) β-D-fructo-2,6-
2
pyranose 4.09-4.04 (m, 1H, H6a); 4.04-4.02 (m, 1H, H5); 3.93 (dd, J=10.0Hz, J=3.5Hz, 1H, H4);
3
.83 (d, J=10.0Hz, 1H, H3); 3.76-3.72 (m, 2H, H6b, H1a); 3.60 (d, J=11.7Hz, 1H, H1b), β-D-
fructo-2,5-furanose 4.17-4.13 (m, 2H, H3, H4); 3.84 (m, 1H, H5); 3.83 (m, 1H, H6a); 3.69 (m, 1H,
H6b); 3.63 (d, J=12.1Hz, 1H, H1a); 3.58 (d, J=12.1Hz, 1H, H1b), α-D-fructo-2,5-furanose 4.17-
4
3
.13 (m, 2H, H3, H5); 4.05-4.01 (m, 1H, H4); 3.86-3.82 (m, 1H, H6a); 3.74-3.70 (m, 1H, H6b);
1
3
.72-3.68 (m, 2H,H1a/b). C NMR (101 MHz, D O): δ (ppm) β-D-fructo-2,6-pyranose 99.12
2
(C2); 70.72 (C4); 70.25 (C5); 68.61 (C3); 64.94 (C1); 64.42 (C6), β-D-fructo-2,5-furanose 102.54
(C2); 81.72 (C5); 76.44 (C3); 75.49 (C4); 63.74 (C1); 63.43 (C6), α-D-fructo-2,5-furanose 105.49
(
C2); 83.0 (C3); 82.37 (C5); 77.09 (C4); 63.96 (C1); 62.15 (C6). m/z HRMS found [M+Cl]-
215.0317, C H O Cl requires 215.0321.
6
12
6
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ASSOCIATED CONTENT
Supporting Information.
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Production of DTA, DAAO and TK , H NMR, C NMR spectra of ketoses 3 and 4.
Rg
gst
AUTHOR INFORMATION
Corresponding Authors
* Laurence.hecquet@uca.fr
*
Juliette.martin@seqens.com
ACKNOWLEDGMENTS
Funding Sources
This work was funded by the Fonds Régional Innovation Laboratoire (grant
DOS00494484/00, to L.H.).
REFERENCES
1
.
R. N. Patel, Ed. Biocatalysis in the Pharmaceutical and Biotechnology Industries, CRC Press, 2010.
2. a) N. J. Turner E. O’Reilly, Biocatalytic retrosynthesis, Nat. Chem. Biol., 2013, 9, 285-288. b) R. A.
Sheldon, J. M. Woodley, Role of Biocatalysis in Sustainable Chemistry Chem. Rev., 2018, 118, 801–
838.
3
.
J. H. SchrittwieSer, S. Velikogne, M. Hall W. Kroutil, Artificial Biocatalytic Linear Cascades for
Preparation of Organic Molecules Chem. Rev., 2018, 118, 270–348.
25
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Organic Process Research & Development
Page 26 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.
a) P. Clapes, W.-D. Fessner, G. Sprenger, A. K. Samland, Curr. Opin. Chem. Biol., 2010, 14, 154-167.
b) M. Müller, D. Gocke, M. Pohl, Thiamin diphosphate in biological chemistry: exploitation of diverse
thiamin diphosphate‐dependent enzymes for asymmetric chemoenzymatic synthesis, FEBS J., 2009,
11, 2894-2904.
5
6
.
.
K. Fesko, Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced
substrate and stereo specificities, Appl. Microbiol. Biotechnol., 2016, 100, 2579–2590
J. Kim, J. P. Kershner, Y. Novikov, R. K. Shoemaker, S. D. Copley. Three serendipitous pathways in
E. coli can bypass a block in pyridoxal-5′-phosphate synthesis, Mol. Syst. Biol. 2010, 6, 436, 1-13.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7. J.Q. Liu, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, H. Yamada. A novel metal-activated pyridoxal
enzyme with a unique primary structure, low specificity D-threonine aldolase from Arthrobacter sp.
strain DK-38. Molecular cloning and cofactor characterization, J. Biol. Chem. 1998, 273, 16678–
1
6685
8
.
S. E. Franz, J. D. Stewart. Threonine aldolases. Adv Appl. Microbiol. 2014, 88, 57–101.
9. K. Fesko, M. Uhl, J. Steinreiber, K. Gruber, H. Griengl, Biocatalytic access to α,α-dialkyl-α-amino
acids by a mechanism-based approach, Angew, Chem. Int. Ed. 2010, 49, 121–124.
10. a) Z. Li, Y. Gao, H. Nakanishi, X. Gao, L. Cai, Biosynthesis of rare hexoses using microorganisms
and related enzymes, J. Org. Chem. 2013, 9, 2434–2445. b) K. Izumori, Izumoring: A strategy for
bioproduction of all hexoses, J. Biotechnol., 2006, 124, 717–722. c) L. Wen, K. Huang, M. Wei, J.
Meisner, Y. Liu, K. Garner, L. Zang, X. Wang, X. Li, J. Fang, H. Zhang, P.G. Wang, Facile Enzymatic
Synthesis of Ketoses. Angew. Chem. Int. Ed. Engl., 2015, 54, 12654-12658
11. a) Y. Kobori, D. C. Myles, G. M. Whitesides, Substrate specificity and carbohydrate synthesis using
transketolase, J. Org. Chem., 1992, 57, 5899-5907. b) T. Ziegler, A. Straub, F. Effenberger,
Enzym‐katalysierte Synthese von 1‐Desoxymannojirimycin, 1‐Desoxynojirimycin und
1
,4‐Didesoxy‐1,4‐imino‐D‐arabinitol, Angew. Chem. Int. Ed. Engl., 1988, 100, 737–738. c) N. J.
Turner, Applications of transketolases in organic synthesis. Curr. Opin. Biotechnol.,2000, 11, 527–
531. d) Galman, J. L.; Steadmann, D.; Bacon, S.; Morris, P.; Smith M. E. B.; Ward, J.; Dalby, P. A.;
and Hailes, H. C. α,α′-Dihydroxyketone formation using aromatic and heteroaromatic aldehydes with
evolved transketolase enzymes, Chem. Commun. 2010, 46, 7608-7610. e) A. Ranoux,;S. K Karmee,
J. Jin,; A. Bhaduri, A. Caiazzo, I. W. Arends, and U. Hanefeld, Enhancement of the substrate scope
of transketolase, ChemBioChem 2012, 13, 1921-1931.
1
2 F. Dickens, D. H. Williamson, The preparation and properties of lithium hydroxypyruvate and
hydroxypyruvic acid, Biochem. J. 1958, 68, 74-81.
13 J. Abdoul Zabar, I. Sorel, V. Hélaine, F. Charmantray, T. Devamani, D. Yi V. de Berardinis, D. Louis,
P. Marlière, W.D. Fessner, L. Hecquet, Thermostable Transketolase from Geobacillus
26
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Page 27 of 28
Organic Process Research & Development
1
2
3
4
5
6
7
8
9
stearothermophilus: Characterization and Catalytic Properties, Adv. Synth. Catal., 2013, 355, 116-
128.
1
4 a) L. Hecquet, J. Bolte and C. Demuynck, Enzymatic Enzymatic synthesis of “natural-labeled” 6-
deoxy-L-sorbose precursor of an important food flavour, Tetrahedron, 1996, 52, 8223-823. b) M.
Lorillière, M. De Sousa, F. Bruna, E. Heuson, F. Gefflaut, V de Berardinis, T. Saravanan, D. Yi,
W.-D. Fessner, F. Charmantray, L. Hecquet, One-pot, two-step cascade synthesis of naturally rare L-
erythro (3S,4S) ketoses by coupling a thermostable transaminase and transketolase, Green Chem.,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
017, 19, 425-435. c) F. Subrizi, M. Cárdenas-Fernández, G. J. Lye, J. M. Ward, P. A. Dalby, T. D.
Sheppard, H. C. Hailes, Transketolase catalyzed upgrading of L-arabinose: the one-step
stereoselective synthesis of L-gluco-heptulose. Green Chem., 2016, 18, 3158-3165. d) M. Bawn, F.
.
Subrizi, G. J. Lye, T. D. Sheppard, H. C. Hailes, J. M. Ward , One-pot, two-step transaminase and
transketolase synthesis of l-gluco-heptulose from L-arabinose, Enz. Microb. Technol., 2018, 116, 16-
2
2.
1
5 M. L’enfant, F. Bruna, M. Lorillière, N. Ocal, W.-D. Fessner, L. Pollegioni, F. Charmantray, L.
Hecquet, One‐Pot Cascade Synthesis of (3S)‐Hydroxyketones Catalyzed by Transketolase via
Hydroxypyruvate Generated inꢀSitu from D‐Serine by D‐Amino Acid Oxidase, Adv. Synth. Catal.,
2
019, 361, 2550-2558.
1
6 S. Fantinato, L. Pollegioni., S. M. Pilone, Engineering, expression and purification of a His-tagged
chimeric D-amino acid oxidase from Rhodotorula gracilis, Enz. Microb. Technol., 2001, 29, 407–412.
7 K. Jung, M. Seifert, T. Herrling, J. Fuchs, UV-generated free radicals (FR) in skin : their prevention
by sunscreens and their induction by self-tanning agents, Spectrochim. Acta A Mol Biomol. Spectrosc.,
1
2
008, 69, 1423–1428.
1
1
2
8 H. D. Belitz, W. Grosch, P. Schieberle, in Food Chemistry, 2009, 248-270, Springer-Verlag.
9 Protéus by Seqens has a collection of >300 off-the-shelf enzymes
0 H. Charmolue, R. W. Rousseau, L‐serine obtained by methanol addition in batch crystallization,
AIChE J., 1991, 37, 1121-1128
2
1 H. Ikeda, Y. Yonetani, S. I. Hashimoto, Process for producing D-serine, US 7,186,532 B2, 10.05.2002.
22 a) L.Pollegioni, G. Molla, S.Sacchi, E. Rosini, R. Verga, M. S. Pilone, Properties and applications of
microbial D-amino acid oxidases : current state and perspectives, Appl. Microbiol. Biotechnol., 2008,
7
8, 1–16. b) L. Pollegioni, P. Motta, G. Molla, Structure-Function Relationships in L-Amino Acid
Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes. Appl.
Micr. Biotech., 2013, 97, 9323-9341.
23 a) L.Pollegioni, G. Molla, S.Sacchi, E. Rosini, R. Verga, M. S. Pilone, Properties and applications of
microbial D-amino acid oxidases: current state and perspectives, Appl. Microbiol. Biotechnol., 2008,
27
ACS Paragon Plus Environment

Organic Process Research & Development
Page 28 of 28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
8, 1–16. b) L. Pollegioni, P. Motta, G. Molla, Appl. Micr. Biotech. 2013, 97, 9323-9341. b) L.
Pollegioni, K. Diederichs, G. Molla, S. Umhau, W. Welte, S. Ghisla, M. S. Pilone, Structure-Function
Relationships in L-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of
Biotechnologically Relevant Enzymes, J. Mol. Biol. 2002, 324, 535–546. c) L Pollegioni, S Iametti, D
Fessas, L Caldinelli, L Piubelli, A Barbiroli, M.S. Pilone, F. Bonomi, Contribution of the dimeric state
to the thermal stability of the flavoprotein D-amino acid oxidase, Protein Sci., 2003, 12, 1018-1029.
4 G. Ali, T. Moreau, C. Forano, C. Mousty, V. Prevot, F. Charmantray, L. Hecquet, Stereoselective
Synthesis of Ketoses by One-Pot, Three-Step Cascade Reactions for in situ Generation of
Transketolase Substrates by Transaminase and Aldolase, ChemCatChem, 2015, 7, 3163–3170.
5 a) J. Abdoul Zabar, M. Lorillière, D. Yi, L. Nauton, F. Charmantray, V. Hélaine, W.-D. Fessner, L.
Hecquet, Thermostable Transketolase Immobilized on Layered Double Hydroxides in Ionic liquids,
Adv. Syn. Catal. 2015, 357, 1715-1720. b) D. Yi, T. Saravanan, T. Devamani, F. Charmantray, L.
Hecquet and W.-D. Fessner, A thermostable transketolase evolved for aliphatic aldehyde acceptors,
Chem. Commun. 2015, 51, 480-483. c) T. Saravanan, S. Junker, M. Kickstein, J. Hegen, S. Hein, M.
K. Link, S. Witt, M. Lorillière, L. Hecquet, W.-D. Fessner, Complementation of 1-Deoxy-D-xylulose-
5-phosphate Synthase Activity, Angew. Chem. Int. Ed., 2017, 56, 5358-5362. d) M. Lorillière, R.
Dumoulin, M. L’Enfant, A. Rambourdin, V. Thery, L. Nauton, W.-D. Fessner, F. Charmantray, L.
Hecquet, Evolved Thermostable Transketolase for Stereoselective two-carbon Elongation of non-
phosphorylated Aldoses to Naturally Rare Ketoses, ACS Catalysis, 2019, 9, 4754-4763.
6 U.K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4,
Nature, 1970, 227, 680-685.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
2
7 L. Hecquet, J. Bolte, and C. Demuynck, New Assays for Transketolase, Biosci. Biotech. Biochem.,
1993, 57 , 2174-2176.
8 K. Benaissi, V. Hélaine, V. Prévot, C. Forano, L. Hecquet, Efficient Immobilization of Yeast
Transketolase on Layered Double Hydroxides and Application for Ketose Synthesis, Adv. Syn. Catal.,
2
011, 353, 1497–1509.
2
9 M. E. B. Smith, E. G Hibbert, A. B. Jones, P. A. Dalby, H. C. Hailes, Enhancing and Reversing the
Stereoselectivity of Escherichia coli Transketolase via Single-Point Mutations, Adv. Syn. Catal. 2008,
3
50, 2631–2638.
3
3
0 W. Funcke, Detection of the open-chain forms of D-fructose and L-sorbose in aqueous solution by
1
3
using C-n.m.r. spectroscopy, Carbohydr. Res 1979, 75, 305–309.
1 T. Barclay, M. Ginic-Markovic, M. R. Johnston, P. Cooper and N. Petrovsky, Observation of the keto
1
tautomer of D-fructose in D O using H NMR spectroscopy, Carbohydr. Res. 2012, 347, 136–141.
2
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