Synthesis of D-sorbose and D-psicose by recombinant Escherichia coli

Article abstract of DOI:10.1080/07328303.2015.1068794

In the current work, the in vitro synthetic system for rare sugars was successfully transformed into an engineered Escherichia coli with a plasmid containing both the aldolase RhaD and phosphatase YqaB. By taking advantage of the inherent biosynthetic pathways in E. coli, this approach permits the use of simple and cheap glycerol for the synthesis of DHAP in vivo. Moreover, the introduction of the phosphatase into the E. coli system allows for the removal of the phosphate group on the synthetic intermediate to yield the neutral rare sugars, which can be readily secreted to the medium without accumulation in the cell.

Full text of DOI:10.1080/07328303.2015.1068794

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Synthesis of D-Sorbose and D-Psicose by
Recombinant Escherichia coli
Zijie Lia, Beibei Hea, Yahui Gaob & Li Caic
a Key Laboratory of Carbohydrate Chemistry and Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University,
Wuxi, 214122, China
b School of Food Science and Technology, Jiangnan University, Wuxi,
214122, China
c Division of Mathematics and Science, University of South Carolina
Salkehatchie, Walterboro, SC 29488, USA
Published online: 29 Jul 2015.
Click for updates
To cite this article: Zijie Li, Beibei He, Yahui Gao & Li Cai (2015) Synthesis of D-Sorbose and D-
Psicose by Recombinant Escherichia coli, Journal of Carbohydrate Chemistry, 34:6, 349-357, DOI:
10.1080/07328303.2015.1068794
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Journal of Carbohydrate Chemistry, 34:349–357, 2015
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C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1068794
Synthesis of D-Sorbose and
D-Psicose by Recombinant
Escherichia coli
Zijie Li,1 Beibei He,1 Yahui Gao,2 and Li Cai3
1Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of
Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China
2School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
3Division of Mathematics and Science, University of South Carolina Salkehatchie,
Walterboro, SC 29488, USA
GRAPHICAL ABSTRACT
In the current work, the in vitro synthetic system for rare sugars was successfully
transformed into an engineered Escherichia coli with a plasmid containing both the al-
dolase RhaD and phosphatase YqaB. By taking advantage of the inherent biosynthetic
pathways in E. coli, this approach permits the use of simple and cheap glycerol for the
synthesis of DHAP in vivo. Moreover, the introduction of the phosphatase into the E.
coli system allows for the removal of the phosphate group on the synthetic intermediate
to yield the neutral rare sugars, which can be readily secreted to the medium without
accumulation in the cell.
Received May 23, 2015; accepted June 30, 2015.
Address correspondence to Li Cai, Division of Mathematics and Science, University of
South Carolina Salkehatchie, Walterboro, SC 29488, USA. E-mail: caili@mailbox.sc.edu
Color versions of one or more of the figures in the article can be found online at www.
tandfonline.com/lcar.
349

Z. Li et al.
350
Keywords Biosynthesis; Biocatalysis; Biotransformations; in vivo synthesis; Rare
sugars; Escherichia coli
INTRODUCTION
Aldolases from carbohydrate metabolism have been used as an important tool
to perform enzymatic carbon-carbon couplings.[1,2] Mild reaction conditions and
stereoselective formation of C C bonds are some of the major advantages for
these biocatalysts.[3–5] Usually aldolases have high specificity for the donor (nu-
cleophile) substrate, which gives rise to the classification of aldolases.[6] Among
the aldolase families, we have been focusing on dihydroxyacetone phosphate
(DHAP)-dependent aldolases in recent years and investigating the stereochem-
istry of these enzymes in in vitro synthesis of rare sugars.[7–11] However, an im-
portant prerequisite for a suitable biocatalysis is that the substrate of the bio-
catalyst should be readily available in sufficient amount and relatively stable.
Thus, the strict specificity for the donor substrate DHAP, a rather expensive
and unstable compound, is disadvantageous for broad application of DHAP-
dependent aldolases.[12,13] Fortunately, DHAP is an important metabolic inter-
mediate that involves various pathways, which has inspired us to develop effi-
cient ways to generate this compound for synthetic purposes. For example, the
major route for glycerol processing in a number of microorganisms provides
DHAP by the oxidation of L-glycerol 3-phosphate catalyzed by glycerol phos-
phate oxidase (GPO).[14] Not only have we followed this route, but also we have
achieved the successful integration of GPO oxidation and aldolization into a
one-pot four-enzyme system in which DHAP could be generated and used in
situ, so that rare sugars could be produced on preparative scales without iso-
lation of the intermediates (Sch. 1).[7–10]
Scheme 1: In vitro one-pot four-enzyme synthesis of rare sugars via DHAP.
Although this one-pot strategy could address the instability and availabil-
ity issues of DHAP to some extent, a few fundamental problems still need to
be considered, such as the assembly of the multienzyme system and the scala-
bility of this approach. Several drawbacks like equilibrium shifts and enzyme
deactivation should also be considered.
Recently, green production from renewable resources via microbial fermen-
tation has attracted increasing attention as microbial transformation has the
advantages of using cheaper catalysts without purification and avoidance of co-
factor recycling in vitro.[15–18] Therefore, we envisioned that both efficiency and

Enzymatic Synthesis of Carbohydrates
351
practicality will be improved if DHAP could be generated from simple achiral
reagents through microbial metabolic pathways and utilized in situ. We report
here a transformation of the multienzymatic system for the synthesis of rare
sugars into engineered E. coli assembly. As a proof-of-concept demonstration,
L-rhamnulose-1-phosphate aldolase (RhaD) and YqaB phosphatase are over-
expressed in E. coli with glycerol as the carbon source, which were assimilated
through the inherent metabolic pathways to provide DHAP for in situ con-
sumption. Meanwhile, we fed the bacteria with the acceptor D-glyceraldehyde
(D-GA) for the production of D-sorbose and D-psicose.
RESULTS AND DISCUSSION
Glycerol has become a very attractive carbon source, as a large surplus of
glycerol is generated inevitably as a by-product during the production of
biodiesel.[19–21] In wild-type E. coli, the process of glycerol dissimilation is as
follows (Sch. 2): first, glycerol is transported into the E. coli cells by glycerol
facilitator; then, glycerol is phosphorylated by glycerol kinase, producing sn-
glycerol 3-phosphate (G3P); in the subsequent step, G3P is converted into
DHAP catalyzed by glycerol 3-phosphate dehydrogenase, a flavoprotein with
molecular oxygen as the electron acceptor.[22] The resulting DHAP is further
metabolized through the glycolytic pathway. This straightforward biosynthetic
route for DHAP from glycerol has already been successfully applied to the syn-
thesis of nonnatural carbohydrates in vitro by Sheldon et al.[23,24] Motivated by
this pathway and previous research, we visualized that the problem of DHAP
synthesis could be solved through its native microbial generation. If in vivo
DHAP generation is further coupled with in situ aldolization reaction cat-
alyzed by aldolase followed by dephosphorylation, large-scale production could
thus be realized by fermentation, and the desired products could be harvested
and isolated from the culture media (Sch. 3).
Scheme 2: Glycerol dissimilation in E. coli.
L-Rhamnulose-1-phosphate aldolase (RhaD) is a class II DHAP-dependent
aldolase that catalyzes the reversible cleavage of L-rhamnulose-1-phosphate
to DHAP and L-lactaldehyde.[25,26] RhaD from E. coli (RhaDE.coli) is a homote-
tramer of the rare C4-symmetric type, containing two molecules of Zn2+ per
enzyme complex, and each subunit is composed of 274 amino acid residues
with a molecular weight of 30,149 Da.[26,27] Interestingly, Fessner et al. first re-
ported in 1991 that RhaDE.coli can also tolerate aldehyde with D-configuration,
such as D-GA, as an acceptor, and we discovered in our previous study that

Z. Li et al.
352
Scheme 3: Production of D-sorbose and D-psicose using engineered E. coli.
RhaDE.coli actually lost its stereoselectivity in the aldolization to simultane-
ously synthesize D-sorbose and D-psicose.[7,28] As no RhaD expression (or at
very low level) was observed under our culture condition, RhaD overexpression
was performed with pBluescript SK-plasmid. It is also necessary to hydrolyze
the phosphate on the C-1–OH of the aldol adduct (Sch. 1) inherent to the use
of DHAP because it was assumed that phosphorylated intermediates from cel-
lular metabolism were efficiently retained by the plasma membrane, leading
to relatively low product yields. Meanwhile, accumulation of such products at
high levels in vivo was assumed to be toxic or, at least, a burden to the cells.[17]
More practically, sugar molecule without a highly polar phosphate group can
be readily secreted into the culture medium, affording an easier product har-
vest process. Acid phosphatase, commonly used in vitro to remove the phos-
phate group of aldol products, is known to operate at a pH optimum of 4.6,
which precludes its application in E. coli. Fortunately, we found that YqaB
phosphatase from E. coli, belonging to the haloacid dehalogenase superfamily,
shows a remarkably broad substrate spectrum, among which the dephospho-
rylation activity toward D-fructose-1-phosphate is the highest.[29] It was also
discovered in our preliminary results that YqaB also showed comparable ac-
tivity toward D-sorbose-1-phosphate, D-psicose-1-phosphate, and L-fructose-1-
phosphate. Most importantly, this enzyme showed high activity under neutral
or physiological conditions as tested by our in vitro reactions. Thus, to carry
out the reaction sequence, the recombinant plasmid pSK-rhaD-yqaB was con-
structed for coexpression of both aldolase RhaD and phosphatase YqaB in E.
coli for the in vivo rare sugar synthesis (Fig. S1).
E. coli DH5α-RY strain containing the plasmid pSK-rhaD-yqaB was used
to carry out the synthesis of D-sorbose and D-psicose (Sch. 3) with glycerol
added to the culture medium. The donor substrate DHAP was generated in this
recombinant E. coli strain from glycerol through native metabolic pathways.

Enzymatic Synthesis of Carbohydrates
353
Figure 1: Monitor the in vivo reaction progress by HPLC.
The acceptor substrate D-GA was supplied by adding into the medium con-
tinuously (discussed below), which could be transported into the cell through
glycerol facilitator.[30] Then, intracellular DHAP was condensed with D-GA cat-
alyzed by overexpressed RhaD to give the aldol product sugar-1-phosphate,
which was subsequently dephosphorylated by YqaB phosphatase to provide
the final products D-sorbose and D-psicose.
In a typical 200-mL fermentation experiment, the consumption of glycerol,
D-GA, and the formation of products were monitored by HPLC (Fig. 1). The
concentration of glycerol decreased steadily as it was constantly assimilated
by the bacteria. It was noticed that, after the first addition at 5 h fermen-
tation, D-GA would be almost consumed within 3 h, so we had to feed the
bacteria D-GA continuously once it was exhausted. During the whole process
of fermentation, 8 mL of D-GA (0.5 M, 2 mL × 4) was added to the medium.
Consistent with our in vitro studies, two rare sugars D-sorbose and D-psicose
were produced simultaneously (Fig. S2). It seemed that the concentration of
D-GA was a very important limiting factor, as the yields of both products in-
creased dramatically accompanied by the addition of D-GA. Interestingly, as
the fermentation proceeded, the concentration of these two sugar products did
not decrease even after we stopped supplying D-GA, evidencing that they were
not consumed/metabolized by the bacteria, which probably do not possess the
enzymes that can utilize these two rare sugars. Toward the end of the fermen-
tation, the culture broth was centrifuged and the products in both the cells
and supernatant were examined. Cells were disrupted by sonication and an-
alyzed by TLC, but no product was detected. Meanwhile, the concentration of
D-sorbose (1.6 g/L) and D-psicose (1.23 g/L) in the supernatant was determined

Z. Li et al.
354
by HPLC (ratio: D-sorbose/D-psicose 1.3:1, Fig. S2). It indicated that D-sorbose
and D-psicose produced were excreted by the bacteria, which enabled an eas-
ier purification process. After purification by silica gel and Bio-Gel P-2 column
chromatography, a mixture of D-psicose and D-sorbose (259 mg) was obtained
and the isolated conversion yield was 36% based on D-GA. Further separa-
tion by Ca2+ ion exchange resin at elevated temperature afforded D-sorbose
(129 mg) and D-psicose (62 mg), which were separated according to their dif-
ferent coordination affinities to Ca2+ [31]
.
To summarize, we established a method for the synthesis of rare sugars in
engineered E. coli by fermentation. The bacteria were transformed with a plas-
mid containing both the aldolase RhaD and phosphatase YqaB. In our process,
the donor substrate DHAP was generated in vivo from cheap and readily avail-
able glycerol through the native metabolic pathways in the host E. coli without
the introduction of additional enzymes, cofactors, or ATP. Significantly, the fi-
nal products could be released to the culture medium, affording a relatively
easy harvest and purification process. The current method could also avoid the
purification of the aldolase and phosphatase utilized in our in vitro studies and
achieve the regeneration of the phosphate. We believe this approach could ulti-
mately contribute to the large-scale synthesis of other carbohydrates and their
derivatives.
EXPERIMENTAL SECTION
Construction of the Recombinant Plasmids pSK-rhaD-yqaB
Primers pSK-rhaD-F and pSK-rhaD-R were used to amplify the gene en-
coding L-rhamnulose-1-phosphate aldolase with E. coli MG1655 as the tem-
plate. The rhaD gene amplified was digested with BamHI and HindIII, then
ligated into pBluescript SK (-) plasmid with the same enzymes digested to gen-
erate plasmid pSK-rhaD (Fig. S1). Primers pSK-yqaB-F and pSK-yqaB-R were
used to amplify the gene encoding YqaB phosphatase with E. coli MG1655 as
the template. The yqaB gene amplified was digested with HindIII and XhoI,
then ligated into pSK-rhaD plasmid with the same enzymes digested to gener-
ate plasmid pSK-rhaD-yqaB (Fig. S1).
Analytical Method
The fermentation progress was monitored by HPLC. Samples were taken
at regular intervals to monitor the consumption of D-GA and production of
D-sorbose and D-psicose. After centrifugation, the supernatant was applied to
HPLC column (Aminex HPX-87H, 300 × 7.8 mm) with 5 mM sulfuric acid as
the mobile phase and detected with the Refractive Index Detector. The flow
rate was 0.5 mL/min, and the column temperature was 60◦C.

Enzymatic Synthesis of Carbohydrates
355
General Procedure for the In Vivo Synthesis and Purification
of D-sorbose and D-psicose
E. coli DH5α was transformed with the plasmid pSK-rhaD-yqaB, result-
ing in the recombinant strain DH5α-RY, which was used to carry out the in
vivo synthesis of D-sorbose and D-psicose. A 4-mL overnight culture of E. coli
DH5α-RY was inoculated to 200 mL fresh LB medium supplemented with
6.5 g/L glycerol and 100 μg/mL ampicillin, and grown aerobically at 37◦C,
225 rpm until the OD600 reached 0.8–1.0 (∼3 h). IPTG was thus added at a
final concentration of 1 mM to induce the coexpression of RhaD and YqaB for
2–3 h. Then D-GA (0.5 M, filter-sterilized stock solution) was added into the
medium with a final concentration of 0.45 g/L. D-GA was added to the medium
continuously when consumed as indicated by HPLC. The pH was adjusted to
7.0 using sodium hydroxide during the fermentation process. Upon completion,
the fermentation broth was centrifuged at 4,000 × g for 20 min at 4◦C. The su-
pernatant was decolored with activated carbon and concentrated with rotary
evaporator under reduced pressure at 40–45◦C. The residue was purified by
flash silica gel column chromatography (EtOAc/iPrOH/H2O 9/3/1 (v/v/v)) to af-
ford the crude products as a yellow syrup, which was further purified by P-2
gel filtration. The mixture of D-sorbose and D-psicose could be separated by
Ca2+ ion exchange resin column (temperature 65◦C, ddH2O as the elutant, flow
rate ∼1.5 mL/min).
FUNDING
This work was supported by the National Natural Science Foundation of
China (21302069), China Postdoctoral Science Foundation (2014M551500),
Key Project of Chinese Ministry of Education (313027), and 111 Project (No.
111-2-06). LC gratefully acknowledges financial support from the University
of South Carolina and USC Salkehatchie.
SUPPLEMENTARY DATA
Supplementary data related to this article can be accessed on the publisher’s
website.
REFERENCES
1. Iturrate, L.; Sanchez-Moreno, I.; Oroz-Guinea, I.; Perez-Gil, J.; Garcia-Junceda,
E. Preparation and characterization of a bifunctional aldolase/kinase enzyme: a more
efficient biocatalyst for C C bond formation. Chemistry 2010, 16, 4018–4030.
2. Machajewski, T.D.; Wong, C.H. The catalytic asymmetric aldol reaction. Angew.
Chem., Int. Ed. Engl. 2000, 39, 1352–1374.

Z. Li et al.
356
3. Samland, A.K.; Sprenger, G.A. Microbial aldolases as C-C bonding
enzymes–unknown treasures and new developments. Appl. Microbiol. Biotechnol.
2006, 71, 253–264.
4. Fessner, W.-D. Enzyme mediated C-C bond formation. Curr. Opin. Chem. Biol.
1998, 2, 85–97.
5. Fessner, W.-D.; Helaine, V. Biocatalytic synthesis of hydroxylated natural products
using aldolases and related enzymes. Curr. Opin. Biotechnol. 2001, 12, 574–586.
6. Fessner, W.-D.; Walter, C. Enzymatic C-C bond formation in asymmetric synthesis.
Top. Curr. Chem. 1997, 184, 97–194.
7. Li, Z.; Cai, L.; Qi, Q.; Wang, P.G. Enzymatic synthesis of D-sorbose and D-psicose
with aldolase RhaD: effect of acceptor configuration on enzyme stereoselectivity. Bioorg.
Med. Chem. Lett. 2011, 21, 7081–7084.
8. Li, Z.; Cai, L.; Qi, Q.; Styslinger, T.J.; Zhao, G.; Wang, P.G. Synthesis of rare sugars
with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8. Bioorg.
Med. Chem. Lett. 2011, 21, 5084–5087.
9. Li, Z.; Cai, L.; Wei, M.; Wang, P.G. One-pot four-enzyme synthesis of ketoses with
fructose 1,6-bisphosphate aldolases from Staphylococcus carnosus and rabbit muscle.
Carbohydr. Res. 2012, 357, 143–146.
10. Li, Z.; Qiao, Y.; Cai, L.; Nakanishi, H.; Gao, X.-D. Characterization of glycerol phos-
phate oxidase from Streptococcus pneumoniae and its application for ketose synthesis.
Bioorg. Med. Chem. Lett. 2015, 25, 504–507.
11. Li, Z.; Gao, Y.; Nakanishi, H.; Gao, X.; Cai, L. Biosynthesis of rare hexoses
using microorganisms and related enzymes. Beilstein J. Org. Chem. 2013, 9, 2434–
2445.
12. Breuer, M.; Hauer, B. Carbon-carbon coupling in biotransformation. Curr. Opin.
Biotechnol. 2003, 14, 570–576.
13. Sanchez-Moreno, I.; Garcia-Garcia, J.F.; Bastida, A.; Garcia-Junceda, E. Mul-
tienzyme system for dihydroxyacetone phosphate-dependent aldolase catalyzed C-C
bond formation from dihydroxyacetone. Chem. Commun. (Cambridge, U. K.) 2004,
1634–1635.
14. Schumperli, M.; Pellaux, R.; Panke, S. Chemical and enzymatic routes to dihy-
droxyacetone phosphate. Appl. Microbiol. Biotechnol. 2007, 75, 33–45.
15. Ajikumar, P.K.; Xiao, W.H.; Tyo, K.E.; Wang, Y.; Simeon, F.; Leonard, E.; Mucha,
O.; Phon, T.H.; Pfeifer, B.; Stephanopoulos, G. Isoprenoid pathway optimization for
Taxol precursor overproduction in Escherichia coli. Science 2010, 330, 70–74.
16. Causey, T.B.; Shanmugam, K.T.; Yomano, L.P.; Ingram, L.O. Engineering Es-
cherichia coli for efficient conversion of glucose to pyruvate. Proc. Natl. Acad. Sci. U.
S. A. 2004, 101, 2235–2240.
17. Popp, A.; Nguyen, H.T.; Boulahya, K.; Bideaux, C.; Alfenore, S.; Guillouet, S.E.;
Nevoigt, E. Fermentative production of L-glycerol 3-phosphate utilizing a Saccha-
romyces cerevisiae strain with an engineered glycerol biosynthetic pathway. Biotechnol.
Bioeng. 2008, 100, 497–505.
18. Yang, J.; Zhu, Y.; Li, J.; Men, Y.; Sun, Y.; Ma, Y. Biosynthesis of rare ketoses
through constructing a recombination pathway in an engineered Corynebacterium glu-
tamicum. Biotechnol. Bioeng. 2015, 112, 168–180.
19. Pagliaro, M.; Ciriminna, R.; Kimura, H.; Rossi, M.; Della Pina, C. From glycerol to
value-added products. Angew. Chem., Int. Ed. Engl. 2007, 46, 4434–4440.

Enzymatic Synthesis of Carbohydrates
357
20. Trinh, C.T.; Srienc, F. Metabolic engineering of Escherichia coli for efficient con-
version of glycerol to ethanol. Appl. Environ. Microbiol. 2009, 75, 6696–6705.
21. da Silva, G.P.; Mack, M.; Contiero, J. Glycerol: a promising and abundant carbon
source for industrial microbiology. Biotechnol. Adv. 2009, 27, 30–39.
22. Truniger, V.; Boos, W. Mapping and cloning of gldA, the structural gene of the
Escherichia coli glycerol dehydrogenase. J. Bacteriol. 1994, 176, 1796–1800.
23. Schoevaart, R.; van Rantwijk, F.; Sheldon, R.A. Carbohydrates from glycerol:
an enzymatic four-step, one-pot synthesis. Chem. Commun. (Cambridge, U. K.) 1999,
2465–2466.
24. Schoevaart, R.; van Rantwijk, F.; Sheldon, R.A. A four-step enzymatic cascade for
the one-pot synthesis of non-natural carbohydrates from glycerol. J. Org. Chem. 2000,
65, 6940–6943.
25. Sugiyama, M.; Hong, Z.; Greenberg, W.A.; Wong, C.H. In vivo selection for the
directed evolution of L-rhamnulose aldolase from L-rhamnulose-1-phosphate aldolase
(RhaD). Bioorg. Med. Chem. 2007, 15, 5905–5911.
26. Kroemer, M.; Merkel, I.; Schulz, G.E. Structure and catalytic mechanism of l-
rhamnulose-1-phosphate aldolase. Biochemistry 2003, 42, 10560–10568.
27. Grueninger, D.; Schulz, G.E. Antenna domain mobility and enzymatic reaction of
l-rhamnulose-1-phosphate aldolase. Biochemistry 2008, 47, 607–614.
28. Fessner, W.-D.; Sinerius, G.; Schneider, A.; Dreyer, M.; Schulz, G.E.; Badia, J.;
Aguilar, J. Diastereoselective enzymatic aldol additions: L-rhamnulose and L-fuculose
1-phosphate aldolases from E. coli. Angew. Chem., Int. Ed. Engl. 1991, 30, 555–558.
29. 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. Genome-wide analysis of substrate specificities
of the Escherichia coli haloacid dehalogenase-like phosphatase family. J. Biol. Chem.
2006, 281, 36149–36161.
30. Heller, K.B.; Lin, E.C.; Wilson, T.H. Substrate specificity and transport properties
of the glycerol facilitator of Escherichia coli. J. Bacteriol. 1980, 144, 274–278.
31. Huang, W.; Mu, W.; Jiang, B. Study on separation and purification of D-tagatose.
Food Ferment. Ind. 2008, 34, 168–171.