Transforming flask reaction into cell-based synthesis: Production of polyhydroxylated molecules via engineered Escherichia coli

Article abstract of DOI:10.1021/acscatal.5b00953

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been intensively studied and widely used in the synthesis of carbohydrates and complex polyhydroxylated molecules. However, strict specificity toward donor substrate DHAP greatly hampers their synthetic utility. Here, we transformed DHAP-dependent aldolases-mediated by in vitro reactions into bioengineered Escherichia coli (E. coli). Such flask-to-cell transformation addressed several key issues plaguing in vitro enzymatic synthesis: (1) it solves the problem of DHAP availability by in vivo-hijacking DHAP from the glycolysis pathway of the bacterial system, (2) it circumvents purification of recombinant aldolases and phosphatase, and (3) it dephosphorylates the resultant aldol adducts in vivo, thus eliminating the additional step for phosphate removal and achieving in vivo phosphate recycling. The engineered E. coli strains tolerate a wide variety of aldehydes as acceptor and provide a set of biologically relevant polyhydroxylated molecules in gram scale.

Full text of DOI:10.1021/acscatal.5b00953

Letter
pubs.acs.org/acscatalysis
Transforming Flask Reaction into Cell-Based Synthesis: Production of
Polyhydroxylated Molecules via Engineered Escherichia coli
Mohui Wei,†,⊥ Zijie Li,‡,⊥ Tiehai Li,† Baolin Wu,† Yunpeng Liu,† Jingyao Qu,† Xu Li,† Lei Li,† Li Cai,§
and Peng George Wang*,†,∥
†Center for Diagnostics and Therapeutics, Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, United States
‡The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan
University, Wuxi 214122, China
§Department of Chemistry, University of South Carolina Salkehatchie, Walterboro, South Carolina 29488, United States
∥State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300457, China
S
* Supporting Information
ABSTRACT: Dihydroxyacetone phosphate (DHAP)-depend-
ent aldolases have been intensively studied and widely used in
the synthesis of carbohydrates and complex polyhydroxylated
molecules. However, strict specificity toward donor substrate
DHAP greatly hampers their synthetic utility. Here, we
transformed DHAP-dependent aldolases-mediated by in vitro
reactions into bioengineered Escherichia coli (E. coli). Such flask-
to-cell transformation addressed several key issues plaguing in
vitro enzymatic synthesis: (1) it solves the problem of DHAP
availability by in vivo-hijacking DHAP from the glycolysis
pathway of the bacterial system, (2) it circumvents purification of recombinant aldolases and phosphatase, and (3) it
dephosphorylates the resultant aldol adducts in vivo, thus eliminating the additional step for phosphate removal and achieving in
vivo phosphate recycling. The engineered E. coli strains tolerate a wide variety of aldehydes as acceptor and provide a set of
biologically relevant polyhydroxylated molecules in gram scale.
KEYWORDS: DHAP-dependent aldolase, synthetic biology, metabolic engineering, E. coli synthetic factory, polyhydroxylated molecules
ldolases exhibit unrivaled efficiency in the synthesis of
Apolyhydroxylated molecules and β-hydroxy-α-amino
acids,1−4 which are difficult to prepare and handle by
conventional chemical synthesis. Among known aldolases,
DHAP-dependent aldolases have been intensively studied and
widely used in the synthesis of carbohydrates and complex
polyhydroxylated molecules,5−18 because configurations of two
newly generated stereogenic centers can be chosen and
controlled by an appropriate choice of four known DHAP-
Figure 1. DHAP-dependent aldolase-mediated aldol reaction.
dependent aldolases.19 Fructose-1,6-bisphosphate aldolase
(FruA) provides a 3S,4R configuration product; fuculose-1-
phosphate aldolase (FucA) provides a 3R,4R configuration
product; tagatose-1,6-bisphosphate aldolase (TagA) provides a
3S,4S configuration product; rhamnulose-1-phosphate aldolase
(RhuA) provides a 3R,4S configuration product. Therefore, a
complete set of four aldol products could be provided (Figure
1).
However, the strict specificity toward donor substrate DHAP
greatly hampers their synthetic utility because of the high cost
and lability of DHAP.3,20 Therefore, effective production of
DHAP is instrumental, and several chemical and enzymatic
approaches have been developed for its synthesis. Chemical
approaches focus on producing storable precursors that can be
easily converted to DHAP immediately before its use;5,21−28
however, they suffer from low yields, complicated workup, or
toxic reagents or catalysts.29 Enzymatic approaches generate
DHAP in situ and follow three general routes: phosphorylation
of dihydroxyacetone (DHA),14,30−34 oxidation of glycerol 3-
phosphate,35−39 and mimicking glycolysis.5,40 Although enzy-
matic approaches start from cheap nonphosphorylated
precursors (DHA, glycerol, sucrose), they employ multiple
costly isolated enzymes (Scheme 1). Both types of approaches
require further improvement to serve as a basis for scalable and
Received: May 6, 2015
Revised: May 27, 2015
© XXXX American Chemical Society
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Letter
a
machinery” to execute transformations inside cells has
completely revolutionized conventional enzymatic synthe-
sis.51−53 Advances in DNA technologies and bioinformatics
enable the reconstruction and perfection of such genetic
devices. Thus, synthetic biology would provide appealing
opportunities and solutions to the aforementioned issues by
technically manipulating microbial hosts to execute the
aldolase-catalyzed reactions inside cells and afford the desired
products.
Scheme 1. Principal Enzymatic Routes to DHAP
Here, we report the transformation of DHAP-dependent
aldolase-mediated in vitro reactions into engineered E. coli for
facile and effective production of polyhydroxylated molecules
(Scheme 2c). In glycolysis, glucose is metabolized into fructose
1,6-bisphosphate via three enzymatic steps and is then split by
FruA into two interconvertible triose phosphates, DHAP and D-
glyceraldehyde-3-phosphate (GAP).54 The concentration ratio
of DHAP to GAP is 96% to 4% because of the favored
formation of DHAP by TIM.55 We envision that by introducing
and overexpressing an aldolase gene and a phosphatase gene in
E. coli cells, overexpressed aldolase can hijack DHAP from the
glycolytic pathway and couple it with exogenous aldehyde to
provide phosphorylated aldol adduct, which is dephosphory-
lated by overexpressed phosphatase and released from the host
cell to give the desired product (Scheme 3).
a
DHAK, dihydroxyacetone kinase; GK, glycerol kinase; AK, acetate
kinase; PK, pyruvate kinase; GPO, L-glycerol 3-phosphate oxidase; XI,
xylose isomerase; HK, hexokinase; GI, glucose 6-phosphate isomerase;
FPK, fructose 6-phosphate kinase; TIM, triose phosphate isomerase;
PEP, phosphoenolpyruvate; and FBP, fructose 1,6-biphosphate.
cost-effective production of DHAP. Another issue associated
with synthetic utility of DHAP-dependent aldolases is that an
additional step is required to remove phosphate group of aldol
adducts (Scheme 2a).
Scheme 3. Hijacking DHAP from the Glycolytic Pathway
Scheme 2. DHAP-Dependent Aldolase-Mediated Aldol
Reaction
A desirable solution is to eliminate the requirement of DHAP
and use readily available, inexpensive DHA. Recent efforts in
overcoming DHAP-dependence by directed evolution of
aldolases, reaction/substrate engineering, and exploitation of
newly discovered enzymes have achieved great success.
Fructose 6-phosphate aldolase (FSA)20,41−44 and engineered
transaldolase B45−47 can accept DHA, hydroxyacetone (HA),
and hydroxylbutone (HB) as donor substrate. FSA even can
accept glycolaldehyde as a donor substrate.43 In addition,
engineered RhuA48 or wild type RhuA with the presence of
borate buffer49,50 also can accept DHA as a donor substrate;
however, these DHAP-independent methods can provide only
3S,4R and 3R,4S configuration products (Scheme 2b). Access
to 3R,4R and 3S,4S products still requires DHAP as the donor,
and to the best of our knowledge, none of these methods has
ever been realized on more than lab scale. Therefore,
developing a general method to solve the above-mentioned
issues is greatly desired.
To fulfill such a transformation, three major issues need to be
addressed. First, a suitable aldolase gene must be introduced
and overexpressed in the host cell. The expressed aldolase
would couple the glycolysis pathway with the aldol reaction to
afford phosphorylated aldol adducts. Second, an appropriate
phosphatase gene needs to be introduced and overexpressed.
High intracellular accumulation of phosphorylated aldol
adducts is expected to be toxic or, at least, a burden to cells.
The phosphatase should be able to selectively dephosphorylate
the resultant aldol adducts under physiological conditions but
without any interference to other phosphorylated metabolic
intermediates, which may have a negative influence on the
whole engineered system. After dephosphorylation, the final
product would be secreted out of host cells. This will shift
glycolysis and the aldol reaction toward product formation and
make purification of the final product much easier. In addition,
the removed phosphate could be recycled inside cells. Third,
bacteria growth and product synthesis must be well balanced.
Hijacking DHAP from glycolysis will greatly reduce energy
production, disrupt the redox balance, and reduce growth in
Synthetic biology, a recently emerging discipline that utilizes
elaborate bioengineered organisms as “programmable synthetic
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Letter
host cells. A suitable condition must be explored to maximize
production while maintaining bacteria growth.
Table 1. Condition Optimization for Maximum Production
We are very interested in FruA, one class of DHAP-
dependent aldolase, owing to its high stereoselectivity and
relaxed aldehyde substrate specificity. FruA from Staphylococcus
carnosus (FruAS.car) encoded by gene fda displays unusual
stability across a wide range of temperature and pH conditions
while retaining quite relaxed acceptor specificity.56,57 At the
same time, FruAS.car has been demonstrated to exhibit high
enantioselectivity in aldol reactions, selectively furnishing aldol
products with a 3S,4R-threo configuration.57 Thus, we chose
FruAS.car as the aldolase. YqaB from E. coli (YqaBE. coli) encoded
by gene yqaB could dephosphorylate D-fructose-1-phosphate
but shows no activity toward aldose phosphates, ketose-
terminal phosphates, or other phosphorylated metabolic
intermediates.58 Our in vitro study also showed that YqaBE. coli
could dephosphorylate D-sorbose-1-phosphate, D-psicose-1-
phosphate, L-tagatose-1-phosphate, and L-fructose-1-phosphate
but showed almost no activity toward DHAP under neutral
conditions. Therefore, we chose YqaBE. coli as the phosphatase.
Meanwhile, we chose 3-trifluoroacetamido propanal 1 as the
model aldehyde acceptor owing to its excellent solubility in
water.
To construct an recombinant E. coli strain, fda and yqaB
genes were cloned into pCDFDuet-1 (Novagen), a vector that
allows high-level expression of two proteins under induction of
isopropyl β-D-1-thiogalactopyranoside (IPTG). The resulting
plasmid pCDF-fda-Y was transformed into E. coli BL21Star
(DE3) to provide recombinant E. coli strain FruA-Y, which was
grown aerobically at 37 °C, 220 rpm in LB broth medium until
its OD600 value reached 1.0. Then the temperature was
switched to 30 °C, and IPTG was added to induce coexpression
of FruA and YqaB for 12 h. Subsequently, 1 and glucose were
fed. To our delight, after purification, 234 mg of 2 was provided
with 11.3% yield.
After validation of such transformation, we set out to
optimize the fermentation conditions by using 1 as a model
acceptor. Several key parameters critical to fermentation yield
were investigated (Table 1). Glucose is the energy source and
carbon source of living cell systems, and a high concentration of
glucose will lead to catabolite repression. To determine the
optimal initial glucose concentration in a medium, a variety of
glucose concentrations, ranging from 2 to 12 g/L, were
examined (entries 1−6). After a glucose concentration was over
the 4 g/L threshold, the yield of 2 would not increase further
and remained almost constant until it reached 12 g/L, at which
point the yield of 2 began to decrease. Therefore, the initial
glucose concentration was chosen to be 4 g/L. Notably, if
glucose was fed during the cell proliferation phase or induction
stage, the yield of 2 would decrease dramatically.
cell
density
OD600
c
glucose
b
incubation
time (h)
yield
d
a
entry
medium
LB Broth
(g/L)
(%)
1
2
2
4
1.0
1.0
12
12
12
12
12
12
11
10
9
19.7
27.6
27.5
27.6
27.4
15.4
30.1
33.5
36.7
41.7
41.7
49.8
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
LB Broth
3
6
1.0
4
8
1.0
5
10
12
4
1.0
6
1.0
7
1.2
8
4
1.4
9
4
1.6
10
11
12
4
1.8
8
4
1.95
>1.8
8
LB Broth/ECAM
(3/1, v/v)
4
8
13
14
15
LB Broth/ECAM
(1/1, v/v)
4
4
>1.8
>1.8
8
8
50.0
40.4
LB Broth/ECAM
(1/3, v/v)
ECAM
4
4
>1.8
>1.8
11
8
28.5
0
e
16
LB Broth/ECAM
(1/1, v/v)
a
Fermentation conditions: strain FruA-Y was grown aerobically at 37
°C, 220 rpm in 200 mL medium until OD600 reached certain value,
then the temperature was switched to 30 °C, and IPTG was added to
induce coexpression of FruA and YqaB for 12 h. Subsequently, 8 mmol
1 and glucose were fed. Initial glucose concentration in medium. At
this cell density, IPTG was added. HPLC yield based on acceptor 1.
b
c
d
e
E. coli strain BL21Star (DE3) was used.
synthesize, although ECAM (see recipe in Supporting
Information) is a mineral salt medium and provides all kinds
of metals and essential trace elements for E. coli. Thus, to probe
the effect of the culture medium on the output of the E. coli
strain, LB Broth and ECAM media were examined, as well as
their combinations. As demonstrated in Table 1, entries 11−15,
LB Broth turned out to be far superior to ECAM with respect
to yield of 2 (entry 11 vs 15); however, an appropriate
combination of these two media can increase the yield of 2.
When these two media were mixed in a 3:1 volumetric ratio,
the yield of 2 was increased from 41.7 to 49.8% (entry 11 vs
entry 12). The yield of 2 was further increased to 50.0% by
mixing LB Broth and ECAM in a 1:1 volumetric ratio (entry
13).
A further increase in the ECAM ratio led to a decrease in the
production yield (entry 14). Therefore, the optimal medium is
a combination of LB Broth and ECAM in a 1:1 volumetric
ratio. In addition, when E. coli strain BL21Star (DE3) was used
to perform fermentation under optimized conditions, no
product was detected (entry 16), which indicated that the
background reactions were negligible.
Addition of IPTG would affect the growth of cells and
expression of the target genes. Therefore, IPTG was added at
different levels of cell density, determined by corresponding
OD600 values, to investigate the effect of IPTG addition time
point on fermentation yield. After 12 h of induction, the OD600
value of all cell cultures reached around 2.3. As illustrated in
entries 2 and 7−11, the yield of 2 increased in an almost linear
relationship with the increase of the cell OD600 values until the
OD600 equaled 1.8, at which point the yield of 2 reached a
plateau at 41.7%. Thus, the optimum time point of IPTG
addition is the time at which the OD600 value is ≥1.8.
After we identified the optimal fermentation conditions, we
set out to investigate the scope and limitations of such a FruA-Y
E. coli synthetic factory by using a variety of cell-membrane-
permeable aldehydes as acceptors. The FruA-Y strain
demonstrated superb promiscuity toward different aldehyde
acceptors. As indicated in Table 2, a set of small aldehydes were
taken up by FruA-Y E. coli cells and subjected to the aldol
reaction with glycolytic intermediate DHAP, followed by in situ
LB Broth medium is a nutritionally rich medium and supplies
essential growth factors that E. coli would otherwise have to
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D-Fagomine, 1-deoxymannojirimycin (DMJ), 1-deoxynojir-
imycin (DNJ), and their derivatives are effective inhibitors of
glycosidases and glycosyltransferases and exert a profound
effect on N-linked glycoprotein processing and maturation, as
well as cell−cell and cell−virus recognition.59 Therefore, they
have gained considerable clinical importance in the treatment
of cancer, type II diabetes, viral diseases such as HIV, hepatitis
B and C, Gaucher’s disease, and other glycosphingolipid storage
disorders.60−63 For example, D-fagomine can effectively reduce
the blood glucose peak when taken together with sucrose or
starch, without stimulating insulin release and can help to
eliminate the excess of enterobacteria and lower weight gain by
selectively agglutinating fimbriated enterobacteria and inhibit-
ing their adhesion to the intestinal mucosa.64,65 Miglitol, N-
hydroxyethyl DNJ, is currently used as a potent second-
generation digestive α-glucosidase inhibitor for treatment of
type II diabetes; Miglustat, N-butyl DNJ, is currently used for
treatment of type 1 Gaucher disease (GD1) and Niemann−
Pick type C (NPC) disease.66−68 By simple deprotection and
reductive amination, product 2, 3, and 4 were transformed to D-
fagomine, DMJ and DNJ respectively with high yields, and no
other diastereomers were detected by 1H NMR analysis
(Scheme 4).
Table 2. Production of Polyhydroxylated Molecules via E.
coli Strain FruA-Y
Scheme 4. Synthesis of D-Fagomine, DMJ, and DNJ
a
Fermentation conditions: strain FruA-Y was grown aerobically at 37
°C, 220 rpm in 1 L LB Broth/ECAM (1/1, v/v) medium until the
OD600 reached 1.8−2.0, then the temperature was switched to 30 °C,
and IPTG was added to induce coexpression of FruA and YqaB for 12
h. Subsequently, the aldehyde acceptor and glucose were fed.
b
c
1
Determined by H NMR. HPLC yield based on aldehyde acceptor.
d
e
f
60 mmol aldehyde was used. 70 mmol aldehyde was used. 1 L of
ECAM was used as the medium and 20 mmol of aldehyde was used.
g
h
Determined by HPLC. 27.3 mmol aldehyde was used. ND, not
determined.
dephosphorylation, to yield the desired aldol products. When 1
was used as acceptor, 2.47 g of 2 was provided with 23.8% yield
as a single stereoisomer (dr > 95:5). However, concentration of
2 in the fermentation medium was 5.18 g/L (determined by
HPLC), and the corresponding yield was 50.0% (entry 1). The
low isolated yield is mainly attributed to loss of product during
purification. Along the same line, 3-trichloroacetamido
propanal gave 4.41 g of (3S,4R)-6-trichloroacetamido-1,3,4-
trihydroxyhexan-2-one with 35.7% yield and 92:8 dr (entry 2);
3-difluoroacetamido propanal gave 2.26 g of (3S,4R)-6-
difluoroacetamido-1,3,4-trihydroxyhexan-2-one with 23.4%
yield and 92:8 dr (entry 3); 3-(methylthio)propanal gave 327
mg of (3S,4R)-1,3,4-trihydroxy-6-(methylthio)hexan-2-one
with 2.8% yield and 92:8 dr (entry 4); and 4,4,4-
trifluorobutanal gave 716 mg of (3S,4R)-7,7,7-trifluoro-1,3,4-
trihydroxyheptan-2-one with 4.7% yield and 87:13 dr (entry 5).
Low fermentation yields of 3-(methylthio)propanal and 4,4,4-
trifluorobutanal are mainly due to vaporization of aldehyde
acceptors during incubation and their poor cell membrane
permeability. When D-glyceraldehyde was used as the acceptor,
947 mg of D-fructose was afforded with 26.3% yield; however,
the HPLC yield before purification was 65.6% (entry 6). In
addition, when (R)-3-trifluoroacetamido-2-hydroxypropanal
and (S)-3-trifluoroacetamido-2-hydroxypropanal were used as
acceptors, 827 mg of 3 and 513 mg of 4 were afforded (entries
7−8).
To further expand applications of such transformation,
recombinant E. coli strains FucA-Y and RhuA-Y were
constructed following the same procotol of FruA-Y with the
fucA gene encoding L-fuculose-1-phosphate aldolase (FucA)
from Thermus thermophilus HB8 and rhuA gene encoding L-
rhamnulose-1-phosphate aldolase (RhuA) from E. coli. For the
FucA-Y strain, D-glyceraldehyde gave 960 mg of D-psicose with
35.6% yield and 92:8 dr (Table 3, entry 1), and 1 gave 665 mg
of (3R,4R)-6-trifluoroacetamido-1,3,4-trihydroxyhexan-2-one 5
with 12.8% yield and 87:13 dr (Table 3, entry 2). For the
RhuA-Y strain, D-glyceraldehyde provided 281 mg of D-piscose
and 286 mg of D-sorbose with 10.4% and 10.6% yields,
respectively (Table 3, entry 3); 1 yielded 1.16 g of (3R,4S)-6-
trifluoroacetamido-1,3,4-trihydroxyhexan-2-one 6 with 22.4%
yield and 89:11 dr (Table 3, entry 4). The successful
application of D-glyceraldehyde and 1 in strains FucA-Y and
RhuA-Y indicated that many other aldehydes also could be
applied in these two E. coli strains.
FruA, FucA, and RhuA also were used to synthesize 2, 5, and
6 in vitro via a one-pot, four-enzyme system (Table 4). GPO
catalyzed the in situ generation of DHAP from DL-glycerol 3-
phosphate, then aldolases coupled DHAP with aldehyde 1 to
give the phosphorylated aldol adducts, which were dephos-
phorylated by acid phosphatase (AP) to give the aldol products
2, 5, and 6. FruA gave 2 exculsively (dr > 95:5, entry 1), FucA
provided 5 with 87:13 dr (entry 2), and RhuA yielded 6 with
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In conclusion, we have successfully transformed DHAP-
dependent aldolases mediated in vitro reactions into engineered
E. coli for practical and effective production of polyhydroxylated
molecules. Such a transformation solves the problem of DHAP
availability by hijacking the DHAP from the glycolytic pathway,
circumvents purification of recombinant aldolases and
phosphatase, dephosphorylates the aldol adducts in vivo, and
recycles phosphate inside E. coli cells. Operational simplicity,
low cost, and easy scalability of fermentation shows such a
transformation holds enormous value in the synthesis of
biologically relevant polyhydroxylated molecules on an
industrial scale. Efforts on enhancing production efficiency by
optimizing E. coli strains via bioengineering and simplifying
purification procedures are ongoing.
Table 3. Production of Polyhydroxylated Molecules via E.
coli Strains FucA-Y and RhuA-Y
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscatal.5b00953.
Experimental procedure, characterization data for all new
compounds, selected NMR spectra (PDF)
a
b
Fermentation conditions: same as Table 2. 1 L of ECAM was used
c
as the medium and 15 mmol of aldehyde was used. 1 L of LB Broth/
ECAM (1/1, v/v) was used as the medium, and 20 mmol of aldehyde
was used. Determined by HPLC. Determined by H NMR.
d
e
1
AUTHOR INFORMATION
Corresponding Author
*E-mail: pwang11@gsu.edu.
■
Table 4. One-Pot Four-Enzyme Synthesis of 2, 5, and 6
Author Contributions
⊥M.W. and Z.L. contributed equally to this work.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
This work was supported by the NIH (GM085267, AI083754).
Dr. Z. Li is grateful for support from the National Natural
Science Foundation of China (21302069).
REFERENCES
■
(1) Fesko, K.; Gruber-Khadjawi, M. ChemCatChem 2013, 5, 1248−
1272.
(2) Clapes, P.; Garrabou, X. Adv. Synth. Catal. 2011, 353, 2263−
2283.
(3) Brovetto, M.; Gamenara, D.; Saenz Mendez, P.; Seoane, G. A.
Chem. Rev. 2011, 111, 4346−4403.
(4) Clapes, P.; Fessner, W.-D.; Sprenger, G. A.; Samland, A. K. Curr.
Opin. Chem. Biol. 2010, 14, 154−167.
a
1
Determined by H NMR.
(5) Bednarski, M. D.; Simon, E. S.; Bischofberger, N.; Fessner, W. D.;
Kim, M. J.; Lees, W.; Saito, T.; Waldmann, H.; Whitesides, G. M. J.
Am. Chem. Soc. 1989, 111, 627−635.
89:11 dr (entry 3). The stereoselectivities of FruA, FucA, and
RhuA are consistent with our in vivo results (Table 2, entry 1;
Table 3, entry 2 and 4); therefore, these engineered E. coli
strains can serve as a general and effective method for the
practical production of polyhydroxylated molecules.
(6) Fessner, W.-D.; Sinerius, G.; Schneider, A.; Dreyer, M.; Schulz, G.
E.; Badia, J.; Aguilar, J. Angew. Chem., Int. Ed. Engl. 1991, 30, 555−558.
(7) Espelt, L.; Parella, T.; Bujons, J.; Solans, C.; Joglar, J.; Delgado,
A.; Clapes, P. Chem. - Eur. J. 2003, 9, 4887−4899.
(8) Espelt, L.; Bujons, J.; Parella, T.; Calveras, J.; Joglar, J.; Delgado,
A.; Clapes, P. Chem. - Eur. J. 2005, 11, 1392−1401.
(9) Moris-Varas, F.; Qian, X.-H.; Wong, C.-H. J. Am. Chem. Soc.
1996, 118, 7647−7652.
(10) Calveras, J.; Egido-Gabas, M.; Gomez, L.; Casas, J.; Parella, T.;
Joglar, J.; Bujons, J.; Clapes, P. Chem. - Eur. J. 2009, 15, 7310−7328.
(11) Calveras, J.; Bujons, J.; Parella, T.; Crehuet, R.; Espelt, L.; Joglar,
J.; Clapes, P. Tetrahedron 2006, 62, 2648−2656.
To confirm that the C1, C2, C3 carbons of products were
derived from supplemented glucose via DHAP of the glycolytic
pathway, FruA-Y was fed with [U-13C6] glucose as the sole
carbon source and 1 as the aldehyde acceptor. Fermentation
was carried out using the same protocol as described previously
by using citric acid-free ECAM as the culture medium. After
1
purification, the product was characterized by H and 13C
NMR, which indicated that the C1, C2, and C3 carbons truly
came from [U-13C6] glucose as integrals of the three carbons
are much stronger than other carbons as a result of 100% 13C
abundance (see Supporting Information).
(12) El Blidi, L.; Assaf, Z.; Camps Bres, F.; Veschambre, H.; Thery,
V.; Bolte, J.; Lemaire, M. ChemCatChem 2009, 1, 463−471.
(13) Zannetti, M. T.; Walter, C.; Knorst, M.; Fessner, W.-D. Chem. -
Eur. J. 1999, 5, 1882−1890.
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(14) Wong, C. H.; Whitesides, G. M. J. Org. Chem. 1983, 48, 3199−
3205.
(15) Laborda, P.; Sayago, F. J.; Cativiela, C.; Parella, T.; Joglar, J.;
(46) Schneider, S.; Gutierrez, M.; Sandalova, T.; Schneider, G.;
Clapes, P.; Sprenger, G. A.; Samland, A. K. ChemBioChem 2010, 11,
681−690.
(47) Rale, M.; Schneider, S.; Sprenger, G. A.; Samland, A. K.;
Fessner, W.-D. Chem. - Eur. J. 2011, 17, 2623−2632.
(48) Garrabou, X.; Joglar, J.; Parella, T.; Crehuet, R.; Bujons, J.;
Clapes, P. Adv. Synth. Catal. 2011, 353, 89−99.
(49) Sugiyama, M.; Hong, Z.; Whalen, L. J.; Greenberg, W. A.;
Wong, C.-H. Adv. Synth. Catal. 2006, 348, 2555−2559.
(50) Garrabou, X.; Calveras, J.; Joglar, J.; Parella, T.; Bujons, J.;
Clapes, P. Org. Biomol. Chem. 2011, 9, 8430−8436.
(51) Khalil, A. S.; Collins, J. J. Nat. Rev. Genet. 2010, 11, 367−379.
(52) Endy, D. Nature 2005, 438, 449−453.
Clapes, P. Org. Lett. 2014, 16, 1422−1425.
(16) Concia, A. L.; Gomez, L.; Parella, T.; Joglar, J.; Clapes, P. J. Org.
Chem. 2014, 79, 5386−5389.
(17) Fessner, W.-D.; Eyrisch, O. Angew. Chem., Int. Ed. Engl. 1992,
31, 56−58.
(18) Li, Z.; Cai, L.; Wei, M.; Wang, P. G. Carbohydr. Res. 2012, 357,
143−146.
(19) Machajewski, T. D.; Wong, C. H. Angew. Chem., Int. Ed. 2000,
39, 1352−1375.
(20) Sugiyama, M.; Hong, Z.; Liang, P.-H.; Dean, S. M.; Whalen, L.
J.; Greenberg, W. A.; Wong, C.-H. J. Am. Chem. Soc. 2007, 129,
14811−14817.
(53) Purnick, P. E. M.; Weiss, R. Nat. Rev. Mol. Cell Biol. 2009, 10,
410−422.
(54) Romano, A. H.; Conway, T. Res. Microbiol. 1996, 147, 448−455.
(55) Harris, T. K.; Cole, R. N.; Comer, F. I.; Mildvan, A. S.
Biochemistry 1998, 37, 16828−16838.
(21) Colbran, R. L.; Jones, J. K. N.; Matheson, N. K.; Rozema, I.
Carbohydr. Res. 1967, 4, 355−358.
(22) Pederson, R. L.; Esker, J.; Wong, C. H. Tetrahedron 1991, 47,
2643−2648.
(56) Brockamp, H. P.; Kula, M. R. Appl. Microbiol. Biotechnol. 1990,
34, 287−291.
(23) Jung, S.-H.; Jeong, J.-H.; Miller, P.; Wong, C.-H. J. Org. Chem.
1994, 59, 7182−7184.
(57) Brockamp, H. P.; Kula, M. R. Tetrahedron Lett. 1990, 31, 7123−
7126.
(24) Gefflaut, T.; Lemaire, M.; Valentin, M.-L.; Bolte, J. J. Org. Chem.
1997, 62, 5920−5922.
(58) Kuznetsova, E.; Proudfoot, M.; Gonzalez, C. F.; Brown, G.;
Omelchenko, M. V.; Borozan, I.; Carmel, L.; Wolf, Y. I.; Mori, H.;
Savchenko, A. V.; Arrowsmith, C. H.; Koonin, E. V.; Edwards, A. M.;
Yakunin, A. F. J. Biol. Chem. 2006, 281, 36149−36161.
(59) Wardrop, D. J.; Waidyarachchi, S. L. Nat. Prod. Rep. 2010, 27,
1431−1468.
(25) Ferroni, E. L.; DiTella, V.; Ghanayem, N.; Jeske, R.; Jodlowski,
C.; O’Connell, M.; Styrsky, J.; Svoboda, R.; Venkataraman, A.;
Winkler, B. M. J. Org. Chem. 1999, 64, 4943−4945.
(26) Meyer, O.; Rohmer, M.; Grosdemange-Billiard, C. Tetrahedron
Lett. 2004, 45, 7921−7923.
(60) Asano, N. Curr. Top. Med. Chem. 2003, 3, 471−484.
(61) Asano, N. Glycobiology 2003, 13, 93−104.
(27) Charmantray, F.; El Blidi, L.; Gefflaut, T.; Hecquet, L.; Bolte, J.;
Lemaire, M. J. Org. Chem. 2004, 69, 9310−9312.
(28) Meyer, O.; Ponaire, S.; Rohmer, M.; Grosdemange-Billiard, C.
Org. Lett. 2006, 8, 4347−4350.
(29) Schuemperli, M.; Pellaux, R.; Panke, S. Appl. Microbiol.
Biotechnol. 2007, 75, 33−45.
(30) Crans, D. C.; Whitesides, G. M. J. Am. Chem. Soc. 1985, 107,
7019−7027.
(31) Yanase, H.; Okuda, M.; Kita, K.; Sato, Y.; Shibata, K.; Sakai, Y.;
Kato, N. Appl. Microbiol. Biotechnol. 1995, 43, 228−234.
(32) Itoh, N.; Tujibata, Y.; Liu, J. Q. Appl. Microbiol. Biotechnol. 1999,
51, 193−200.
(62) Robina, I.; Moreno-Vargas, A. J.; Carmona, A. T.; Vogel, P.
Curr. Drug Metab. 2004, 5, 329−361.
(63) Dwek, R. A.; Butters, T. D.; Platt, F. M.; Zitzmann, N. Nat. Rev.
Drug Discovery 2002, 1, 65−75.
(64) Gomez, L.; Molinar-Toribio, E.; Calvo-Torras, M. A.;
Adelantado, C.; Juan, M. E.; Planas, J. M.; Canas, X.; Lozano, C.;
Pumarola, S.; Clapes, P.; Torres, J. L. Br. J. Nutr. 2012, 107, 1739−
1746.
(65) Ramos-Romero, S.; Molinar-Toribio, E.; Gomez, L.; Perez-
Jimenez, J.; Casado, M.; Clapes, P.; Pina, B.; Torres, J. L. Obesity 2014,
22, 976−979.
(33) Sanchez-Moreno, I.; Francisco Garcia-Garcia, J.; Bastida, A.;
Garcia-Junceda, E. Chem. Commun. 2004, 1634−1635.
(34) Sanchez-Moreno, I.; Iturrate, L.; Doyagueez, E. G.; Martinez, J.
A.; Fernandez-Mayoralas, A.; Garcia-Junceda, E. Adv. Synth. Catal.
2009, 351, 2967−2975.
(66) Horne, G.; Wilson, F. X.; Tinsley, J.; Williams, D. H.; Storer, R.
Drug Discovery Today 2011, 16, 107−118.
(67) Nash, R. J.; Kato, A.; Yu, C. Y.; Fleet, G. W. Future Med. Chem.
2011, 3, 1513−1521.
(68) Zhang, Z.-X.; Wu, B.; Wang, B.; Li, T.-H.; Zhang, P.-F.; Guo, L.-
N.; Wang, W.-j.; Zhao, W.; Wang, P. G. Tetrahedron Lett. 2011, 52,
3802−3804.
(35) Fessner, W. D.; Sinerius, G. Angew. Chem., Int. Ed. Engl. 1994,
33, 209−212.
(36) Schoevaart, R.; van Rantwijk, F.; Sheldon, R. A. J. Org. Chem.
2000, 65, 6940−6943.
(37) Hettwer, J.; Oldenburg, H.; Flaschel, E. J. Mol. Catal. B: Enzym.
2002, 19−20, 215−222.
(38) Charmantray, F.; Dellis, P.; Samreth, S.; Hecquet, L.
Tetrahedron Lett. 2006, 47, 3261−3263.
(39) Li, Z.; Cai, L.; Qi, Q.; Styslinger, T. J.; Zhao, G.; Wang, P. G.
Bioorg. Med. Chem. Lett. 2011, 21, 5084−5087.
(40) Fessner, W.-D.; Walter, C. Angew. Chem., Int. Ed. Engl. 1992, 31,
614−616.
(41) Schurmann, M.; Sprenger, G. A. J. Biol. Chem. 2001, 276,
11055−11061.
(42) Castillo, J. A.; Calveras, J.; Casas, J.; Mitjans, M.; Vinardell, M.
P.; Parella, T.; Inoue, T.; Sprenger, G. A.; Joglar, J.; Clapes, P. Org.
Lett. 2006, 8, 6067−6070.
(43) Garrabou, X.; Castillo, J. A.; Guerard-Helaine, C.; Parella, T.;
Joglar, J.; Lemaire, M.; Clapes, P. Angew. Chem., Int. Ed. 2009, 48,
5521−5525.
(44) Concia, A. L.; Lozano, C.; Castillo, J. A.; Parella, T.; Joglar, J.;
Clapes, P. Chem. - Eur. J. 2009, 15, 3808−3816.
(45) Schneider, S.; Sandalova, T.; Schneider, G.; Sprenger, G. A.;
Samland, A. K. J. Biol. Chem. 2008, 283, 30064−30072.
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DOI: 10.1021/acscatal.5b00953
ACS Catal. 2015, 5, 4060−4065