Selective synthesis of 2-furoic acid and 5-hydroxymethyl-2-furancarboxylic acid from bio-based furans by recombinant Escherichia coli cells

Article abstract of DOI:10.1016/j.mcat.2019.03.006

Upgradation of bio-based furans into chemicals and biofuels has received great interest recently. In this work, we reported selective synthesis of furan carboxylic acids from the corresponding aldehydes by recombinant Escherichia coli cells expressing 3-succinoylsemialdehyde-pyridine dehydrogenase (SAPDH) from Comamonas testosteroni SC1588. The effects of induction and reaction conditions on whole-cell catalytic oxidation of furfural (FF) were studied. High temperature induction resulted in decreased activities of recombinant cells, likely due to improper protein folding. Nonetheless, recombinant cells induced under high temperature enable the byproduct furfuryl alcohol to be faster re-oxidized into 2-furoic acid (FCA) than those induced under low temperature. So the yield and selectivity of FCA were improved significantly by using high temperature induction, at expense of slightly longer reaction periods. The activities of recombinant cells highly depended on pH. The tolerant levels of this recombinant strain toward FF and 5-hydroxymethylfurfural (HMF) were approximately 100 mM. FCA and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) were obtained with the yields of 95–98%. FCA of up to 147 mM was produced by a fed-batch strategy, in a quantitative yield. In addition, most aromatic aldehydes tested were transformed into the target carboxylic acids by this biocatalytic method, with the yields up to 100%.

Full text of DOI:10.1016/j.mcat.2019.03.006

MolecularCatalysis469(2019)68–74
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Selective synthesis of 2-furoic acid and 5-hydroxymethyl-2-furancarboxylic
acid from bio-based furans by recombinant Escherichia coli cells
Sai-Sai Shia, Xue-Ying Zhanga, Min-Hua Zonga, Chuan-Fu Wangb, Ning Lia,⁎
a School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
b National Institute of Clean-and-Low-Carbon Energy, Beijing 102211, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Aldehyde dehydrogenases
Biocatalysis
Furans
Platform chemicals
Selective oxidation
Upgradation of bio-based furans into chemicals and biofuels has received great interest recently. In this work, we
reported selective synthesis of furan carboxylic acids from the corresponding aldehydes by recombinant
Escherichia coli cells expressing 3-succinoylsemialdehyde-pyridine dehydrogenase (SAPDH) from Comamonas
testosteroni SC1588. The effects of induction and reaction conditions on whole-cell catalytic oxidation of furfural
(FF) were studied. High temperature induction resulted in decreased activities of recombinant cells, likely due to
improper protein folding. Nonetheless, recombinant cells induced under high temperature enable the byproduct
furfuryl alcohol to be faster re-oxidized into 2-furoic acid (FCA) than those induced under low temperature. So
the yield and selectivity of FCA were improved significantly by using high temperature induction, at expense of
slightly longer reaction periods. The activities of recombinant cells highly depended on pH. The tolerant levels of
this recombinant strain toward FF and 5-hydroxymethylfurfural (HMF) were approximately 100 mM. FCA and 5-
hydroxymethyl-2-furancarboxylic acid (HMFCA) were obtained with the yields of 95–98%. FCA of up to 147 mM
was produced by a fed-batch strategy, in a quantitative yield. In addition, most aromatic aldehydes tested were
transformed into the target carboxylic acids by this biocatalytic method, with the yields up to 100%.
1. Introduction
chemical showed an antitumor activity [11]. Chemical methods are still
playing a predominant role in the synthesis of HMFCA and FCA
Furfural (FF) and 5-hydroxymethylfurfural (HMF) that can be syn-
thesized via carbohydrate dehydration are top value-added platform
chemicals derived from biomass [1]. Recently, upgrading of these
furans into commercially interesting chemicals and biofuels has at-
tracted considerable interest [2–6]. 2-Furoic acid (2-furancarboxylic
acid, FCA) and 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) are
the oxidation products of FF and HMF, respectively. FCA is an im-
portant fine chemical, which has wide applications in pharmaceutical,
agrochemical, flavor, and fragrance industries [2]. Presently, FCA is
industrially produced from FF via Cannizzaro reaction in the presence
of NaOH [2]. However, an equal amount of the byproduct furfuryl al-
cohol forms simultaneously, resulting in the theoretical selectivity of
50%. HMFCA is a promising building block in polymer industry, since it
can not only be directly used for manufacturing polyesters [7], but also
is a key starting material for the synthesis of renewable terephthalic
acid (TPA) [8,9], a monomer in the production of polyethylene ter-
ephthalate (PET) that is widely used in the industrial production of
synthetic fibers and plastic bottles. In addition, HMFCA is a synthon in
the synthesis of an interleukin inhibitor [10]. It was reported that this
nowadays [2,12].
It is well known that furans are susceptible to polymerization and
decomposition under harsh conditions (e.g., high temperature, and the
presence of acids), thus resulting in the formation of the byproducts
[4,13]. And even rapid aging and decomposition of HMF were observed
when storing under room temperature [14]. Hence, developing prac-
tical routes remains challenging for selective and efficient conversion of
furans into the desired products. Biocatalysis may open up novel pos-
sibilities for upgradation of this kind of inherently unstable chemicals
[15,16], because biotransformations are generally performed under
mild reaction conditions using environmentally friendly biocatalysts,
and are exquisitely selective. However, both FF and HMF have a
strongly inhibitory and toxic effect on biocatalysts [17,18], which may
be one of the biggest barriers for the development of practical bioca-
talytic processes. Also, it may account for the fact that there are limited
studies on biocatalytic upgrading of FF and HMF compared to chemical
processes. Krystof et al. reported a chemo-enzymatic route for the
synthesis of FCA and HMFCA [19], in which peracids produced in situ
were capable of selectively oxidizing the formyl group in furans; acids
⁎ Corresponding author.
E-mail address: lining@scut.edu.cn (N. Li).
https://doi.org/10.1016/j.mcat.2019.03.006
Received 14 December 2018; Received in revised form 28 February 2019; Accepted 4 March 2019
2468-8231/©2019ElsevierB.V.Allrightsreserved.

S.-S. Shi, et al.
MolecularCatalysis469(2019)68–74
were obtained in the yields of 80–91%. HMF was quickly oxidized into
HMFCA with a yield of 94% by using xanthine oxidase [20]. Baeyer-
Villiger monooxygenases were also found to enable FF and HMF to be
oxidized into the corresponding acids; the conversions were approxi-
mately 60–85% [21]. Recently, our group exploited an alcohol dehy-
drogenase coupled with an efficient NAD(P)+ in situ regeneration
system for selective synthesis of FCA and HMFCA [22]. In addition to
enzymes, whole cells were also used as catalysts for selective oxidation
of furan aldehydes into carboxylic acids [23–27]. We recently isolated a
HMF-tolerant strain Comamonas testosteroni SC1588 for the synthesis of
HMFCA; the desired product was afforded in a yield of approximately
98% when the substrate concentration was 160 mM [28].
(99%) was bought from ThermoFisher Scientific (Beijing, China). 5-
Methyl-2-furancarboxylic acid (97%) was obtained from Sigma-Aldrich
(USA). Benzyl alcohol (99%) was purchased from Kermel Chemical
Reagent Co., Ltd. (Tianjin, China). Benzoic acid (99.5%) was obtained
from Damao Chemical Reagent Ltd. (Tianjin, China). The gene se-
quence of CtSAPDH, and its cloning and expression in E. coli are
available in supplementary material.
2.2. Cultivation of E. coli_CtSAPDH
The medium for cell cultivation was Luria Bertani (LB) medium
containing 10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract (pH
7.2), which was supplemented with 50 mg/mL kanamycin. E.
coli_CtSAPDH cells were pre-cultivated at 37 °C and 180 r/min for 12 h
in 50 mL LB medium. Then, 1% seed culture was inoculated into the
fresh LB medium containing 50 mg/L kanamycin and incubated at 37 °C
and 180 r/min. When the optical density of the culture at 600 nm
reached 0.6-0.8, isopropyl β-D-thiogalactoside (IPTG) as inducer was
added to the medium at a final concentration of 0.05 mM, and culti-
vation was performed at 30 °C and 160 r/min for 20 h. The cells were
harvested by centrifugation (8000 r/min, 5 min, 4 °C) and washed twice
with 0.85% NaCl solution, followed by re-suspension in phosphate
buffer (0.2 M, pH 7) to give a cell concentration of 50 mg (cell wet
weight) per mL.
Aldehyde dehydrogenases (ALDHs) represent the natural catalysts
for the oxidation of aldehydes in cells. However, previous examples of
synthetic applications of this type of enzymes were scarce [29]. Their
biotransformation applications are not starting until very recently
[25,30,31].
3-Succinoylsemialdehyde-pyridine
dehydrogenase
(SAPDH) is a critical enzyme involved in the catabolism of nicotine,
which catalyzes the oxidation of 3-succinoylsemialdehyde-pyridine into
3-succinoyl-pyridine [32]. Wang et al. constructed a whole-cell bioca-
talyst harboring a SAPDH for the synthesis of 3-succinoyl-pyridine, a
valuable feedstock in the production of hypotensive agents [33]. In this
work, we broadened the catalytic application of SAPDH to the oxidation
of aromatic aldehydes (Scheme 1). A SAPDH responsible for HMF oxi-
dation was identified from C. testosteroni SC1588, and heterologously
expressed in Escherichia coli. Whole-cell catalytic oxidation of FF into
FCA was performed using recombinant E. coli_CtSAPDH. Effect of in-
duction conditions on the catalytic performances of recombinant cells
in FF oxidation was studied. In addition, the biocatalytic process was
optimized to obtain a good yield. A fed-batch strategy was applied for
accumulating the desired product of a high concentration in the reac-
tion mixture. Besides, this biocatalytic approach was exploited for se-
lective oxidation of other aldehydes into the target carboxylic acids.
2.3. General procedure for biocatalytic oxidation of FF
Typically, 4 mL of phosphate buffer (0.2 M, pH 7) containing 50 mM
FF and 50 mg (cell wet weight) per mL microbial cells was incubated at
30 °C and 160 r/min. Aliquots were withdrawn from the reaction mix-
tures at specified time intervals and diluted with the corresponding
mobile phase prior to HPLC analysis. The conversion was defined as the
ratio of the consumed substrate amount to the initial substrate amount
(in mol). The yield was defined as the ratio of the formed product
amount to the theoretical value based on the initial substrate amount
(in mol). The selectivity was defined as the ratio of the formed product
amount to the total amount of all products (in mol). All the experiments
were conducted at least in duplicate, and the values were expressed as
2. Experimental
2.1. Materials
FF (99%), 2,5-bis(hydroxymethyl)furan (BHMF, 98%) and benzal-
dehyde (98.5%) were obtained from Macklin Biochemical Co., Ltd.
(Shanghai, China). HMF (98%), 5-formylfuran-2-furancarboxylic acid
(FFCA, 98%) and 2,5-furandicarboxylic acid (FDCA, 97%) were pur-
chased from J&K Scientific Ltd. (Guangzhou, China). HMFCA (98%)
and 5-methoxymethylfurfural (MMF, 97%) were bought from Adamas
Reagent Ltd. (Shanghai, China). Furfuryl alcohol (98%), FCA (98%), 5-
methylfurfural (97%), 4-fluorobenzaldehyde (98%), 4-fluorobenzoic
acid (98%), 4-fluorobenzyl alcohol (98%), 4-chlorobenzaldehyde
(97%), 4-chlorobenzoic acid (99%), 4-chlorobenzyl alcohol (99%), 4-
bromobenzaldehyde (97%), 4-bromobenzoic acid (98%), 4-bromo-
benzyl alcohol (99%) and 2,5-diformylfuran (DFF, > 98%) were bought
from TCI (Japan). 5-Methyfurfuryl alcohol (98%) was purchased from
Apollo Scientific Ltd. (UK). 5-Methoxymethyl-2-furancarboxylic acid
the means
standard deviations.
2.4. Synthesis of FCA by fed-batch feeding of substrate
The reaction mixture containing 4 mL phosphate buffer (0.2 M, pH
7), 50 mM FF, and 50 mg (cell wet weight)/mL microbial cells was in-
cubated at 30 °C and 160 r/min. When FF was almost used up, FF of
approximately 0.2 mmol was repeatedly supplemented into the reaction
mixture. Two strategies were applied for controlling pH of reaction
mixtures: one was to add CaCO3 (about 2 mol) to reaction mixture at
the beginning, and the other was to add NaHCO3 (approximately
0.2 mol) when supplementing FF. The changes in the concentrations of
various compounds were monitored by HPLC.
2.5. HPLC analysis
The reaction mixtures were analyzed on a Zorbax Eclipse XDB-C18
column (4.6 mm × 250 mm, 5 μm, Agilent, USA) by using a reversed
phase HPLC equipped with a Waters 996 photodiode array detector
(Waters, USA). For the analysis of the reaction mixtures of FF bio-
transformation, the mobile phase was a mixture of acetonitrile/0.4%
(NH4)2SO4 solution with pH 3.5 (10: 90, v/v) at a flow rate of 0.6 mL/
min. The retention times of FCA (maximum absorption wavelength of
245 nm), furfuryl alcohol (216 nm) and FF (278 nm) were 9.2, 14.3 and
16.3 min, respectively. The analytic methods of other compounds and
their retention times are available in supplementary material.
Scheme 1. Whole-cell catalytic oxidation of aromatic aldehydes into carboxylic
acids.
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MolecularCatalysis469(2019)68–74
Fig. 2. Effect of IPTG concentrations (a) and induction temperature (b) on se-
lective oxidation of FF. General reaction conditions: 50 mM FF, 50 mg/mL (wet
weight) microbial cells, 1 mL phosphate buffer (0.2 M, pH 7), 30 °C, 160 r/min,
10 h; (a): The cells induced in the presence of IPTG of 0.05–1 mM at 17 °C for
20 h; (b): The cells induced in the presence of IPTG of 0.05 mM at 15–35 °C for
20 h.
Fig. 1. Progress curve of biocatalytic oxidation of FF. Reaction conditions:
approximately 50 mM FF, 50 mg/mL (wet weight) microbial cells, 1 mL phos-
phate buffer (0.2 M, pH 7), 30 °C, 160 r/min.
3. Results and discussion
(1–5 h) were required to achieve 100% substrate conversions for the
cells induced under 25–35 °C. In addition, a clear trend was observed:
the transformation rates of FF decreased substantially with increasing
induction temperature from 25 to 35 °C (Fig. S3a). Although the bio-
synthesis of heterologous proteins might accelerate at high induction
temperature, improper protein folding occurred easily and thus a large
amount of inactive inclusion body formed [34,35], resulting in the
decreased apparent activities. On the basis of SDS-PAGE analysis (Fig.
S2a), the formation of more inclusion body was indeed found under
higher induction temperature (e.g., 30–35 °C) compared to that under
lower induction temperature. In contrast, the concentrations of soluble
SAPDH appeared to be lower under higher induction temperature than
those under lower induction temperature (Fig. S2b). Also, it might be
verified by the fact that the FCA yields at 0.5 h reduced with increasing
induction temperature (15–20 °C, 59–61%; 25 °C, 44%, 30–35 °C, 36-
31%, Fig. S3b).
Besides, the induction temperature appeared to have an effect on
the expression of reductases/dehydrogenases present in host cells. Its
effect on the apparent reduction activities of recombinant cells had the
same trend as that on the apparent oxidation activities (Fig. S3b and c).
For example, the yields of furfuryl alcohol were 35–41% at 0.5 h with
the cells induced under 15–20 °C, whereas being 27%, 20% and 9%
with those induced under 25, 30 and 35 °C, respectively (Fig. S3c).
More interestingly, it was found that furfuryl alcohol formed was re-
oxidized faster by the cells induced under higher temperature
(30–35 °C) than by those induced under 15–25 °C (Fig. S3c). It indicates
that induction temperature may also affect the expression of enzymes
responsible for the re-oxidation of furfuryl alcohol. To verify our as-
sumption, direct oxidation of furfuryl alcohol was conducted with re-
combinant cells induced under various temperature (Fig. 3). As shown
in Fig. 3, the cells induced under 25–35 °C indeed exhibited much
higher catalytic activities than those induced under 17–20 °C. In addi-
tion to inducer concentrations and induction temperature, induction
time is also one of parameters affecting the enzyme expression. So the
effect of induction periods on the oxidation of FF was investigated when
induction periods varied from 12 to 28 h (Fig. S1). It was observed that
the cells induced for different periods showed similar catalytic perfor-
mances in the oxidation of FF. FCA was produced in the yields of
93–95%, along with trace furfuryl alcohol (< 2%). It suggests that in-
duction periods have no significant influence on the catalytic perfor-
mances of recombinant cells.
3.1. Time course of whole-cell catalytic oxidation of FF
Fig. 1 shows the progress curve of biocatalytic oxidation of FF with
recombinant E. coli expressing CtSAPDH. It was found that recombinant
E. coli_CtSAPDH was capable of efficient transformation of the toxic
substrate FF. The substrate was completely converted within 1 h. On the
contrary, FF of only 7% was transformed when E. coli harboring plain
vector was used as biocatalyst (data not shown). It suggests that
CtSAPDH heterologously expressed in E. coli contributes the transfor-
mation of FF. The desired product FCA of approximately 43 mM (the
yield of 84%) was produced within 1 h (Fig. 1). In addition, furfuryl
alcohol was simultaneously produced as the byproduct, which might
stem from the catalytic behaviors of reductases/dehydrogenases in-
herently present in host cells. Then, furfuryl alcohol was re-oxidized
slowly into FCA, which is the limited step in the synthesis of the desired
product. It is highly consistent with a previous report [28]. The desired
product was furnished in a good yield of 97% after the reaction of 24 h.
3.2. Effect of induction conditions
Induction is crucial for the biosynthesis and folding of heterologous
proteins in recombinant cells, so it may affect the catalytic perfor-
mances of recombinant cells. The influence of induction conditions on
whole-cell catalytic oxidation of FF was studied (Fig. 2 and Fig. S1).
Fig. 2a shows the effect of the concentrations of IPTG, an inducer of the
lactose operon, on the catalytic performances of E. coli cells. Compared
to that in the control, the biocatalytic synthesis of FCA was significantly
improved upon IPTG induction (Fig. 2a), due to the induced expression
of active CtSAPDH (Fig. S2b). However, the concentrations of IPTG had
no significant impact on the catalytic performances of E. coli cells
within the concentration range tested. Comparable substrate conver-
sions as well as similar product yields were observed in all cases
(Fig. 2a).
Interestingly, induction temperature was found to exert a great ef-
fect on the oxidation of FF (Fig. 2b). Microbial cells induced at high
temperature (30 and 35 °C) provided excellent FCA yields (96–99%),
while the FCA yields (79–89%) were lower in other cases. To uncover
the underlying reasons, time courses of FF oxidation catalyzed by whole
cells induced under different temperature were monitored (Fig. S3). It
was found that recombinant cells induced under 15–20 °C were capable
of completely transforming FF in 0.5 h, whereas longer reaction periods
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MolecularCatalysis469(2019)68–74
Fig. 3. Effect of induction temperature on the oxidation of furfuryl alcohol
(solid symbols) into FCA (open symbols). Reaction conditions: 20 mM furfuryl
alcohol, 50 mg/mL (wet weight) microbial cells, 2 mL phosphate buffer (0.2 M,
pH 7), 30 °C, 160 r/min.
Fig. 5. Effect of substrate concentrations on the synthesis of FCA. Reaction
conditions: FF of a designated concentration, 50 mg/mL (wet weight) microbial
cells, 2 mmol CaCO3, 4 mL phosphate buffer (0.2 M, pH 7), 30 °C, 160 r/min.
3.3. Effect of pH and temperature
As shown in Fig. 4b, pH exerted a significant effect on biocatalytic
oxidation of FF. Recombinant cells displayed good catalytic activities
only within a narrow pH range (pH 7–8). Similarly, wild-type C. tes-
tosteroni SC1588 that contains SAPDH also showed narrow pH tolerance
[28]. The substrate conversions were 53–55% at pH 5 and 6, while
being 100% at pH 7 and 8. In addition, significant effect of pH on the
reaction selectivities was observed. FCA selectivities (62–80%) were
unsatisfactory at pH 5–6, while absolute selectivities (100%) were
achieved at pH 7-8. And even, furfuryl alcohol rather than FCA was
produced as the major product at pH 9.
To obtain a good yield, the reaction conditions including reaction
temperature and pH were optimized (Fig. 4). Fig. 4a shows the effect of
reaction temperature on biocatalytic oxidation of FF. The E. co-
li_CtSAPDH cells displayed comparable activities at 20–35 °C, since the
substrate was completely converted within 5 h (data not shown).
However, the substrate conversion was approximately 73% within the
same period when the reaction was performed at 40 °C, despite 95%
conversion in 10 h. It indicates that the reaction temperature as high as
40 °C is unfavorable for transformation of FF by this biocatalyst, likely
due to partial thermal inactivation of enzymes. To identify whether cell
lysis occurred at high temperature, the cell viability was determined
after incubation of 6 h at different temperature in the absence and
presence of FF (Fig. S4). No significant decreases in the cell viability
were observed after 6 h, regardless of whether the substrate is present
or not as well as of temperature. So cell lysis was not a major cause for
the low activity of whole-cell biocatalyst at 40 °C. As shown in Fig. 4a,
the cells displayed the best catalytic performance at 30 °C. The desired
product FCA was achieved in the yield of 95%. More importantly, its
selectivity was up to 100%, since the byproduct furfuryl alcohol was
completely re-oxidized into FCA in 10 h.
3.4. Effect of substrate concentrations
Good substrate tolerance of biocatalysts is of great importance for
their synthetic applications, since high substrate concentrations are
highly desirable for achieving satisfactory productivities in large-scale
production. With the optimal reaction conditions in hand, therefore, the
substrate tolerance of this recombinant strain was evaluated (Fig. 5). It
was found that FF could be smoothly transformed into the desired
product by this biocatalyst when the substrate concentrations were less
than 100 mM. The substrate conversions were up to 100%; FCA was
obtained in excellent yields (96–98%) and absolute selectivities
(100%). However, the substrate conversions decreased to approxi-
mately 74% when the FF concentrations were more than 125 mM (data
not shown). FCA yields were around 55%. These results suggested that
recombinant E. coli_CtSAPDH was able to tolerate FF of up to 100 mM
under the tested reaction conditions. This substrate concentration is
higher than those used in most of the existing biocatalytic processes for
the synthesis of FCA from FF [19,21,24,26,36].
3.5. Synthesis of FCA by a fed-batch strategy
Accumulation of high concentrations of product in the reaction
mixture facilitates its purification. Nevertheless, direct production of
the desired products is usually hard to be achieved from high con-
centrations of such substrates as FF and HMF, due to their significant
toxicity and inhibition against biocatalysts [17,18]. Although the re-
combinant strain was demonstrated to tolerate FF of up to 100 mM
(Fig. 5), the reaction rate was unsatisfactory and a long reaction period
was required for achieving a good yield. Considering the substrate
toxicity and inhibitory effect, a fed-batch strategy in which approxi-
mately 50 mM FF was fed was applied for accumulating a high con-
centration of FCA in the mixture (Fig. 6). The product FCA is a car-
boxylic acid. So pH of the reaction mixtures will decrease with its
Fig. 4. Effect of reaction temperature (a) and pH (b) on selective oxidation of
FF. General reaction conditions: 50 mM FF, 50 mg/mL (wet weight) microbial
cells, 2 mL phosphate buffer (0.2 M, pH 7), 30 °C, 160 r/min, 10 h; (a):
20–40 °C; (b): phosphate buffer (0.2 M, pH 5–8), and Tris−HCl buffer (0.2 M,
pH 9).
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MolecularCatalysis469(2019)68–74
chemical [40]. As shown in Table 1, MMF was found to be an appro-
priate substrate (Entry 6); 5-methoxymethyl-2-furancarboxylic acid, a
feedstock for the synthesis of bio-based TPA [9], was afforded in a 95%
yield. Compared to the wild type strain [28], recombinant cells ex-
hibited much higher catalytic activities in the oxidation of furans in-
cluding FF, DFF and 5-methylfurfural, which may be clearly verified by
the considerably reduced reaction periods. These results may highlight
the superiority of recombinant cells as catalysts to wild-type cells for
biotransformation of these furans. As shown in Table 1, both benzal-
dehyde and its fluorinated derivative were transformed into the target
acids with good yields (90–97%, entries
7 and 8). 4-Chlor-
obenzaldehyde and 4-bromobenzaldehyde were tested with low con-
centrations (Entries 9–11), because of their poor aqueous solubility. 4-
Chlorobenzoic acid was obtained with a 100% yield when the substrate
concentration was 10 mM (Entry 9). When the substrate concentration
was increased to 20 mM under the assistance of dimethyl sulphoxide
(DMSO), unsatisfactory results were obtained (Entries 10 and 11),
which may be partially attributed to the deleterious effect of DMSO on
the cells. To evaluate whether SAPDH has a chiral recognition ability,
kinetic resolution of racemic 2-phenylpropionaldehyde was performed
by using this recombinant strain. Unfortunately, (R)-acid was furnished
with an enantiomeric excess (e.e.) value of approximately 13% (data
not shown). Similarly, ALDHs of other sources displayed poor en-
antioselectivities [25].
Fig. 6. Continuous synthesis of FCA in the presence of CaCO3 (a) and by adding
NaHCO3 (b). General reaction conditions: 50 mM FF, 50 mg/mL (wet weight)
microbial cells, 4 mL phosphate buffer (0.2 M, pH 7), 30 °C, 160 r/min. (a) in
the presence of 2 mmol CaCO3; (b) supplementing NaHCO3 of 0.2 mmol when
feeding FF. Arrows indicate the feed of approximately 0.2 mmol FF.
gradual accumulation, which may exert a negative effect on the activity
of biocatalyst. Indeed, it was found that the final FCA concentration of
only 65 mM was achieved without the addition of bases (Fig. S5). So
two weak bases including CaCO3 and NaHCO3 were supplemented to
control pH of the reaction mixtures. Insoluble CaCO3 was added at the
beginning, while NaHCO3 was simultaneously added with FF feed.
Their effects on biocatalytic oxidation of FF were compared. As shown
in Fig. 6, the substrate transformation became much slower with the
increment of FF feed times, indicating that FF may cause cell damage.
However, the toxic and inhibitory effect of substrate on biocatalyst was
significantly relieved by using a fed-batch strategy, which may be evi-
denced by the reduced reaction period in biotransformation of 100 mM
FF (12 h vs 48 h without FF feed in Fig. 5). As shown in Fig. 6a, FCA was
obtained within 96 h in a quantitative yield (100%) in the presence of
CaCO3. The final concentration of FCA (129 mM) was slightly lower
with the addition of NaHCO3 than that with adding CaCO3 (147 mM).
In addition, furfuryl alcohol produced (around 24 mM) could not be re-
oxidized into FCA (Fig. 6b). It indicates that insoluble CaCO3 seems to
be advantageous over NaHCO3 for the synthesis of FCA, because the
former is able to adjust pH instantaneously, thus reducing the negative
effect of pH change on biocatalyst.
4. Conclusions
A biocatalytic approach was successfully established for selective
oxidation of a series of aromatic aldehydes into the corresponding
carboxylic acids by using recombinant E. coli cells harboring a SAPDH
from C. testosteroni SC1588. This recombinant strain exhibited sa-
tisfactory tolerance toward FF and HMF (up to 100 mM), well-known
potent inhibitors against microorganisms. Of induction conditions ex-
amined, induction temperature was the most significant factor affecting
the catalytic performances of recombinant cells. This recombinant
strain showed good catalytic activities in a narrow pH range (pH 7–8).
So pH control appeared to be of great importance in biocatalytic
synthesis of carboxylic acids using this strain. FCA of up to 147 mM was
synthesized in a fed-batch process, in a quantitative yield. In addition,
this biocatalyst had a broad substrate spectrum, and was capable of
efficiently transforming a variety of furan and benzyl aldehydes into the
target carboxylic acids with good yields. However, this ALDH has no
chiral recognition ability in kinetic resolution of a racemic aldehyde. In
spite of good catalytic performances, some problems associated with
this recombinant biocatalyst have to be addressed in the future. For
example, the long-term stability should be enhanced significantly,
which may be realized by reaction engineering strategies (e.g., cell
immobilization and medium engineering) and protein engineering
[41]. The improved substrate tolerance is highly desired for moving this
green technology forward to the industrial applications.
3.6. Biocatalytic oxidation of aromatic aldehydes
To uncover the substrate spectrum of CtSAPDH, whole-cell catalytic
oxidation of various aromatic aldehydes was performed (Table 1). HMF
was a good substrate for E. coli_CtSAPDH cells. A high yield of HMFCA
(95%) was obtained within 5 h when the substrate concentration was
50 mM (Table 1, entry 1). However, the reaction became very slow
when its concentration increased to 100 mM (Entry 2), although the
yield remained high. With DFF as substrate, FDCA, a promising bio-
based alternative to TPA [37], was produced as the major product
(Entry 3), together with FFCA. However, the substrate conversion (less
than 20%) was pretty poor when the concentration of DFF increased to
50 mM (data not shown), due to great toxicity and inhibition of this
furan dialdehyde against whole-cell biocatalyst. FFCA was quickly
oxidized into FDCA with an excellent conversion (Entry 4). Also, the
desired product 5-methyl-2-furancarboxylic acid was obtained in a
quantitative yield when 5-methylfurfural acted as substrate (Entry 5).
MMF obtained via carbohydrate dehydration in the presence of me-
thanol is an analog of HMF [38,39], but the former has much better
storage stability than the latter. Recently, Carro et al. reported a self-
sustained enzyme cascade for the production of FDCA from this
Declarations of interest
none.
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (21676103), the Natural Science Foundation of
Guangdong Province (2017A030313056), and the Science and
Technology Project of Guangzhou City (201804010179).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2019.03.006.
72

S.-S. Shi, et al.
MolecularCatalysis469(2019)68–74
Table 1
Oxidation of aldehydes into acids using E. coli_CtSAPDH cells.
Entry
Substrate
Product
Concentration (mM)
Time (h)
Conversion (%)
Yield (%)
1
2
50
5
48
100
100
95
95
1
2
100a
3
4
20
50
12
12
100
100
77
1/ 32
1
,
100
5
6
50a
50
5
5
100
100
100
95
7
8
50b
50b
24
24
100
100
97
90
1
0
9
10
11
10
24
24
24
100
83
61
100
59
47
20c
20d
0
0
1
0
Reaction conditions: aldehyde of a designated concentration, 50 mg/mL (wet weight) microbial cells, 4 mL phosphate buffer (0.2 M, pH7.0), 30 °C, 160 r/min.
a
addition of 500 mM CaCO3.
addition of 5% DMSO.
addition of 15% DMSO.
addition of 20% DMSO.
b
c
d
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