Characterization of alditol oxidase from Streptomyces coelicolor and its application in the production of rare sugars

Article abstract of DOI:10.1016/j.bmc.2020.115464

A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor D-glyceraldehyde was directly produced from glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and D-glyceraldehyde was performed by L-rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phosphate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars D-sorbose and D-psicose. To accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldO_S.coe) was expressed in E. coli and the purified AldO_S.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes including AldO_S.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of D-sorbose and D-psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective method for the synthesis of rare sugars.

Full text of DOI:10.1016/j.bmc.2020.115464

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Characterization of alditol oxidase from Streptomyces coelicolor and its
application in the production of rare sugars
Zhou Chen, Zijie Li⁎, Fen Li, Ning Wang, Xiao-Dong Gao⁎
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A synthetic platform for the cascade synthesis of rare sugars using Escherichia coli whole cells was established. In
the cascade, the donor substrate dihydroxyacetone phosphate (DHAP) was generated from glycerol by glycerol
kinase (GK) and glycerol phosphate oxidase (GPO). The acceptor D-glyceraldehyde was directly produced from
glycerol by an alditol oxidase. Then, the aldol reaction between DHAP and D-glyceraldehyde was performed by L-
rhamnulose-1-phosphate aldolase (RhaD) to generate the corresponding sugar-1-phosphate. Finally, the phos-
phate group was removed by fructose-1-phosphatase (YqaB) to obtain the rare sugars D-sorbose and D-psicose. To
accomplish this goal, the alditol oxidase from Streptomyces coelicolor (AldOS.coe) was expressed in E. coli and the
purified AldOS.coe was characterized. Furthermore, a recombinant E. coli strain overexpressing six enzymes in-
cluding AldOS.coe was constructed. Under the optimized conditions, it produced 7.9 g/L of D-sorbose and D-
psicose with a total conversion rate of 17.7% from glycerol. This study provides a useful and cost-effective
method for the synthesis of rare sugars.
Alditol oxidase
Glycerol
Rare sugar
Whole cell
1. Introduction
glycerol via the metabolic pathway, while the acceptor D-glycer-
aldehyde had to be added to the reaction mixture. Since the cost of D-
Rare sugars, which exist rarely in nature, have attracted much at-
tention due to their unique properties.1,2 Among the family of rare
sugars, D-psicose (D-allulose) is the most popular and has great potential
application in the food and pharmaceutical industries.3,4 Moreover, D-
psicose has been approved as “generally regarded as safe” (GRAS) by
the US Food and Drug Administration (FDA). Another rare sugar, D-
sorbose, could inhibit the activity of disaccharidase and suppress the
elevation of postprandial blood levels of glucose.5 The most common
method for rare sugar synthesis is based on the Izumori strategy.6 As the
reaction by epimerase or isomerase is reversible, obtaining the product
with a high yield is much preferred.7–9
glyceraldehyde is relatively high, the conversion of cheap starting
material to D-glyceraldehyde is significant. Although D-glyceraldehyde
production via chemical synthesis has been realized on a large scale, the
chemical route suffers from high cost and tedious steps.19 In contrast,
new short enzymatic routes for the preparation of D-glyceraldehyde are
desirable. It was reported that glyceraldehyde was produced as an in-
termediate for the synthesis of glyceric acid from glycerol by the action
of alditol oxidase from Streptomyces coelicolor A3 (AldOS.coe).20 Up to
now, “true” glycerol oxidases suitable for the synthesis of D-glycer-
aldehyde have not been described yet. Compared with its preferred
substrate sorbitol, AldOS.coe displays lower catalytic activity using gly-
cerol as a substrate.21 In this study, the generation of D-glyceraldehyde
from glycerol by AldOS.coe was characterized. Furthermore, the synth-
esis of D-sorbose and D-psicose only from glycerol was achieved by in-
troducing AldOS.coe into the whole-cell cascade catalysis.
Aldolases have been widely employed for the synthesis of inter-
esting structures, such as thio-sugars, long-chain sugars, and cycli-
tols.10–12 As a complement to the Izumori strategy, aldol addition cat-
alyzed by dihydroxyacetone phosphate (DHAP)-dependent aldolases
provide a very useful method for rare sugar synthesis. In previous study,
a one-pot four-enzyme approach was adopted to synthesize various rare
sugars based on DHAP-dependent aldolases.13–17 More recently, we
established an efficient whole-cell cascade catalysis to produce rare
ketoses from glycerol based on L-rhamnulose-1-phosphate aldolase
(RhaD).18 In our system, the donor substrate DHAP was generated from
2. Results and discussion
2.1. Characterization of the enzymatic properties of AldOS.coe
To perform the characterization of AldOS.coe, this oxidase was
⁎ Corresponding authors.
E-mail addresses: lizijie@jiangnan.edu.cn (Z. Li), xdgao@jiangnan.edu.cn (X.-D. Gao).
https://doi.org/10.1016/j.bmc.2020.115464
Received 26 December 2019; Received in revised form 21 February 2020; Accepted 23 March 2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:ZhouChen,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2020.115464

Z. Chen, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 1. (a) SDS-PAGE analysis of purified AldOS.coe. M: marker, Line 1: purified AldOS.coe; (b) glyceraldehyde was generated by AldOS.coe from glycerol; (c) con-
firmation of D-glyceraldehyde generation by AldOS.coe from glycerol.
Fig. 2. The effects of temperature (a) and pH (b) on the activities of AldOS.coe
.
overexpressed in E. coli Rosetta (DE3) and purified as shown by SDS-
PAGE analysis (Fig. 1a). As an attempt, the oxidative activity of Al-
dOS.coe was determined by high-performance liquid chromatography
(HPLC) analysis. As shown in Fig. 1b, the peak of glyceraldehyde could
be obviously observed, and no glyceric acid was detected. An ex-
planation for undetected glyceric acid was that oxygen supply was
limited under our reaction condition and the reaction time was only
30 min. Although the desired glyceraldehyde was detected and com-
pared with the standard D-glyceraldehyde by HPLC, the configuration of
glyceraldehyde couldn't be confirmed. According to our previous study,
D-sorbose and D-psicose were produced when RhaD tolerated D-glycer-
aldehyde as the acceptor. In contrast, the sole product L-fructose was
generated when RhaD accepted L-glyceraldehyde as the substrate.18
Consequently, we employed AldOS.coe in the synthesis of rare sugars via
one-pot multienzyme reactions. As a result, D-sorbose and
were detected by HPLC suggesting that D-glyceraldehyde was gDe-npesricaotesde
from glycerol by AldOS.coe (Fig. 1c). Next, the optimal reaction condi-
tion of AldOS.coe was further investigated. As illustrated in Fig. 2, the
2

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Scheme 1. Strategy for the whole cell synthesis of D-sorbose and D-psicose only from glycerol.
optimal temperature and pH value for AldOS.coe were 35 °C and 7.5,
respectively. It should be noted that the activity of AldOS.coe decreased
obviously under high temperatures and alkaline pH values.
L) of D-sorbose and D-psicose was achieved using E. coli R-03 whole
cells. The production yield using whole cells of one E. coli strain was
approximately three times that of the mixture of two E. coli strains. The
possible reason could be that more steps involving the output or input
of D-glyceraldehyde were required for the mixture of two types of whole
cells.22
2.2. Validation of the synthetic pathway for production of D-sorbose and D-
psicose from glycerol
2.3. Effect of peroxidase overexpression on the production of rare sugars
To realize whole cell synthesis of D-sorbose and D-psicose only from
the sole carbon source glycerol, the synthetic pathway was constructed
as follows (Scheme 1): the donor substrate DHAP was generated via the
glycerol assimilation pathway, and the endogenous level of DHAP was
enhanced by overexpression of glycerol kinase (GK) and glycerol
phosphate oxidase (GPO). The acceptor D-glyceraldehyde was directly
produced in situ from glycerol by overexpression of the oxidase Al-
dOS.coe. Then, DHAP was coupled with D-glyceraldehyde by RhaD to
generate the corresponding ketose-1-phosphate. Finally, D-sorbose and
D-psicose were produced and released from the cell membrane by re-
moving the phosphate group by fructose-1-phosphatase (YqaB).
To demonstrate the feasibility of producing rare sugars only from
glycerol, E. coli R-01 overexpressing AldOS.coe and E. coli R-02 over-
expressing GK, GPO, RhaD and YqaB (Table 1) were cultured and in-
duced for 20 h at 16 °C, respectively. As a preliminary experiment, the
reaction was carried out using the mixture of E. coli R-01 and E. coli R-
02 whole cells after induction (ratio 1:1) with glycerol as the starting
material. To our delight, the peaks of D-sorbose and D-psicose could be
detected by HPLC using the authentic samples as the standards
(Fig. 3a). To further improve the production yield, the proportions of E.
coli R-01 and E. coli R-02 whole cells were investigated. As illustrated in
Fig. 3b, the total amount of rare sugars (1.5 g/L) was the highest using
E. coli R-01 and E. coli R-02 whole cells at a ratio of 2:1.
As shown in Scheme 1, H2O2 is produced as a byproduct in the
generation of D-glyceraldehyde and DHAP catalyzed by AldOS.coe and
GPO, respectively. Reactive oxygen species (ROS) were formed when
molecular oxygen collided with a variety of flavoenzymes, and high
levels of intracellular ROS led to enzyme inhibition and cell damage.23
It was reported that intracellular H2O2 can be scavenged by endogenous
peroxidases from E. coli, such as Prx02 and KatE.23,24 Inspired by this
result, Prx02 and KatE were overexpressed in E. coli R-03 to obtain E.
coli R-07 and E. coli R-08 (Table 1), respectively. These two strains were
employed for biotransformation using 500 mM glycerol as the carbon
source. To our delight, overexpression of Prx02 or KatE could improve
the total production yield of rare sugars, as shown in Fig. 5. In contrast
to E. coli R-03, the production yield of E. coli R-07 and E. coli R-08 was
approximately 1.7-fold and 1.2-fold at 12 h, respectively. Thus, the E.
coli R-07 strain was selected in the following study.
2.4. Optimization of reaction conditions
To improve the conversion rate of rare sugars, the reaction condi-
tions were further optimized. The pH value of the reaction system had a
significant effect on the catalytic activity, and the optimal pH was 7.5
(Fig. 6a). It should be noted that the relative activity was obviously
decreased at alkaline pH. Then, the influence of temperature on the
production of rare sugars was determined at temperatures ranging from
25 °C to 45 °C, and the optimal temperature was 35 °C (Fig. 6b). Next,
the effect of the whole cell amount was evaluated from 50 to 350 g/L
To simplify the culture process, we attempted to coexpress all en-
zymes in E. coli strain. Moreover, the expression levels of related en-
zymes were optimized using different combinations of plasmids, and
the corresponding strains E. coli R-03, R-04, R-05 and R-06 (Table 1)
were obtained. As shown in Fig. 4, the highest production yield (4.7 g/
Table 1
Strains used in this study.
Strains
Relevant genotype or phenotype
Reference
Invitrogen
E. coli DH5α
endA1 supE44 recA1 gyrA96 relA1 deoR
U169 Φ 80dlacZ ΔM15 mcrAΔ (mrr−
hsdRMS − mcrBC)
E. coli Rosetta (DE3)
Omp Thsd ThsdS (rB-mB-) gal (DE3)
Invitrogen
R-01
R-02
R-03
R-04
R-05
R-06
R-07
R-08
E. coli Rosetta (DE3) containing the plasmid pETDuet-aldO
This study
[18]
E. coli Rosetta (DE3) containing the plasmids pCDFDuet-rhaD-glpO-glpK and pET28a-yqaB
E. coli Rosetta (DE3) containing the plasmids pCDFDuet-rhaD-glpO-glpK and pETDuet-yqaB-aldO
E. coli Rosetta (DE3) containing the plasmids pET28a-rhaD-glpO-glpK and pCDFDuet-yqaB-aldO
E. coli Rosetta (DE3) containing the plasmids pRSFDuet-rhaD-glpO-glpK and pCDFDuet-yqaB-aldO
E. coli Rosetta (DE3) containing the plasmids pRSFDuet-rhaD-glpO-glpK and pETDuet-yqaB-aldO
E. coli Rosetta (DE3) containing the plasmids pCDFDuet-rhaD-glpO-glpK and pETDuet-yqaB-aldO-prx02
E. coli Rosetta (DE3)) containing the plasmids pCDFDuet-rhaD-glpO-glpK and pETDuet-yqaB-aldO-katE
This study
This study
This study
This study
This study
This study
3

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Fig. 3. (a) Whole cell synthesis of rare sugars only from glycerol by HPLC. (b) Optimization of the proportions of whole cells.
glycerol to D-glyceraldehyde. Based on the catalytic property of
AldOS.coe, a synthetic platform using E. coli whole cells containing six
enzymes was established for the production of D-sorbose and D-psicose
only from glycerol. This cascade system was extensively investigated
and optimized for the best result. This strategy could also provide an
efficient alternative approach for the manufacture of other value-added
chemicals from the abundant glycerol.
4. Materials and methods
4.1. Chemicals and materials
D-glyceraldehyde was purchased from Sigma-Aldrich (St. Louis, MO,
USA). PCR product purification kits, isopropyl-β-D-1-thiogalactopyr-
anoside (IPTG), ampicillin, streptomycin and kanamycin were all pur-
chased from Sangon (Shanghai, China). PCR primers and infusion DNA
polymerase were obtained from Tian Lin Co., Ltd (Wuxi, China). D-
sorbose and D-psicose were purchased from TCI (Tokyo, Japan).
Restriction enzymes, Ligation Mix, T4 DNA ligase, One Step Cloning Kit
and In-Fusion HD Cloning kits were purchased from Takara (Japan).
Ca2+ exchange resin was purchased from Suqing Company (Jiangsu,
China). Ni2+-NTA column was purchased from Qiagen (Hilden,
Deutschland). Bio gel P-2 gel, Aminex HPX-87H column
(300 × 7.8 mm) was purchased from Bio-Rad Laboratories, Inc.
(Hercules, CA). All other chemicals were of analytical grade and com-
mercially available.
Fig. 4. Rare sugar synthesis using different E. coli strains.
4.2. Plasmid construction
Bacterial strains, primers and plasmids used in this study are listed
in Table 1, Tables S1 and S2. E. coli DH5α was used for general cloning.
E. coli Rosetta (DE3) was used as host strain for all protein expression
and biocatalysis experiments. LB medium and TB medium were used for
E. coli culture and protein expression, respectively. Ampicillin, strep-
tomycin and kanamycin were used at a concentration of 100, 50 and
50 μg mL−1. The codon-optimized gene of aldO (based on E. coli pre-
ferences) encoding the alditol oxidase from S. coelicolor A3 was syn-
thesized from Genewiz Co., Ltd (Suzhou, China). The gene aldO was
inserted into pET28a plasmid at BamH I and Hind III sites to obtain the
plasmid pET28a-aldO. To construct the plasmid pETDuet-aldO, the aldO
fragment was released from pET28a-aldO after the digestion with Nde I
and Xho I, and this fragment was ligated into the pETDuet-1 plasmid
with the same enzymes digested. The gene yqaB encoding the fructose-
1-phosphatase, prx02 and katE encoding the peroxidase were amplified
from E. coli MG1655 by PCR. The gene yqaB was inserted into the
pETDuet-aldO plasmid at Nco I and EcoR I sites to obtain pETDuet-yqaB-
aldO. The fragments RBS-prx02 and RBS-katE were introduced into
pETDuet-yqaB-aldO at Xho I site using In-Fusion HD Cloning kits to
generate pETDuet-yqaB-aldO-RBS-prx02 and pETDuet-yqaB-aldO-RBS-
Fig. 5. Overexpression of peroxidase improved the production yield.
wet cells. Since the total amount of rare sugars was not increased sig-
nificantly above 150 g/L wet cell, 150 g/L was chosen as the optimal
cell amount (Fig. 6c). Finally, the influence of the initial concentration
of glycerol was evaluated, and 500 mM glycerol was determined as the
best concentration. After that, the production of D-sorbose and D-psicose
was performed only from glycerol using E. coli R-07 whole cells under
the optimized conditions described above. As a result, 7.9 g/L of rare
sugars (D-sorbose/D-psicose = 1.2/1) was produced with a total con-
version rate of 17.7% from glycerol, and the highest volumetric pro-
ductivity was 662 mg/L/h at 36 h (Fig. 7).
3. Conclusion
In summary, AldO from S. coelicolor A3 was capable of converting
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Fig. 6. Effects of pH (a), temperature (b), cell amounts (c) and glycerol concentration (d) on rare sugar production.
Fig. 7. Production of D-sorbose and D-psicose by batch biotransformation from glycerol.
katE, respectively. To construct the plasmid pCDFDuet-yqaB-aldO, the
yqaB-aldO fragment was released from the plasmid pETDuet-yqaB-aldO
after digestion with Xba I and Pac I, and this fragment was ligated to
pCDFDuet-1 using the same enzymes digested. To construct the plasmid
pRSFDuet-rhaD-glpO-RBS-glpK, the fragment rhaD-glpO-RBS-glpK was
released from pCDFDuet-rhaD-glpO-RBS-glpK after digestion with Xba I
and Pac I, and this fragment was introduced into pRSFDuet-1 using the
same enzymes digested. All the recombinant plasmids were confirmed
by sequencing.
centrifugation at 15,000 rpm for 30 min at 4 °C. AldOS.coe was purified
through Ni2+-NTA column (5 mL) and concentrated by ultrafiltration
tube (50 mL). The purity of AldOS.coe was detected by 12% (v/v) SDS-
PAGE and the gels were stained by Coomassie Brilliant Blue.
4.4. Characterization of AldOS.coe
The activity of AldOS.coe was assayed in a reaction mixture con-
taining 50 mM Tris-HCl buffer (pH 7.5), purified AldOS.coe (0.1 mg/mL)
and 10 mM glycerol. After the conversion was performed at 30 °C for
30 min, the formation of glyceraldehyde from glycerol was examined
by HPLC as described below. To confirm the product glyceraldehyde
was indeed D-glyceraldehyde, the purified AldOS.coe was employed in
the one-pot multienzyme reactions for rare sugars synthesis. The reac-
tion mixture (0.2 mL) contained Tris-HCl buffer (50 mM, pH 7.5),
glycerol (20 mM), DL-glycerol-3-phosphate (in excess), GPO (0.01 mg/
mL), RhaD (0.05 mg/mL), YqaB (0.1 mg/mL), AldOS.coe (0.01 mg/mL)
and catalase (0.01 U). This one-pot multienzyme system was performed
at 30 °C for 12 h and the generation of the corresponding rare sugars
was determined by HPLC. To determine the optimal reaction condition
for AldOS.coe, the effects of temperature (25–50 °C) and pH value
4.3. Expression and purification of AldOS.coe
E. coli Rosetta (DE3) carrying the plasmid pET28a-aldO was grown
to an optical density of 0.6–0.8 at 600 nm at 37 °C in LB medium with
50 μg mL−1 kanamycin. Then, 0.1 mM IPTG was added to induce the
expression of AldOS.coe. After the cultivation at 16 °C for 20–24 h, cells
were harvested and washed with 0.9% (wt/vol) NaCl solution by cen-
trifugation at 6000 rpm for 3 min. The cell pellet was subsequently
suspended in the binding buffer (pH 8.0, 50 mM Tris-HCl, 20 mM
imidazole and 150 mM NaCl). Cells were disrupted by sonication for
15 min in an ice bath, and the supernatant was obtained by
5

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(7.0–9.0) on the activities of AldOS.coe were investigated.
Acknowledgements
4.5. Preparation of resting cells
This work was supported by the National Natural Science
Foundation of China (21778023, 21807048), National first-class dis-
cipline program of Light Industry Technology and Engineering
(LITE2018-015), Program of Introducing Talents of Discipline to
Universities (No. 111-2-06).
The recombinant E. coli strain carrying multiple genes was firstly
inoculated in 5 mL LB medium containing appropriate antibiotics at
37 °C for 12 h. Then, the culture was transferred into 100 mL TB
medium in a 500 mL shake flask. When the OD600 of the culture was
0.6–0.8 at 37 °C, IPTG was added with a final concentration of 0.1 mM
to induce the enzyme expression at 16 °C for 20–24 h. The cells were
collected by centrifugation at 3,000 g for 10 min and the cell pellets
were washed by 0.9% (wt/vol) NaCl solution. Then the cells were re-
suspended in an appropriate buffer to desired concentrations as resting
cells for biotransformation.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115464.
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D-psicose was concentrated under reduced
pressure. TDh-esorrbesoisdeueanwd as purified by silica gel chromatography
(EtOAc/iPrOH/H2O 9/3/1 (v/v/v)), which was further separated by
Bio gel P-2 column. Finally, the mixture of D-sorbose and D-psicose was
isolated by a Ca2+ ion-exchange resin at 70 °C. The purified D-sorbose
and D-psicose were identified by 1H NMR.
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by an alditol oxidase and directed evolution for enhanced oxidative activity towards
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
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