Furan Carboxylic Acids Production with High Productivity by Cofactor-engineered Whole-cell Biocatalysts

Article abstract of DOI:10.1002/cctc.202000259

Furan carboxylic acids are useful chemicals in various industries. In this work, biocatalytic production of furan carboxylic acids was reported with high productivities by cofactor-engineered Escherichia coli cells. NADH oxidase (NOX) was introduced into E. coli harboring aldehyde dehydrogenases (ALDHs) to promote intracellular NAD⁺ regeneration, thus significantly enhancing ALDH-catalyzed oxidation. These engineered biocatalysts were capable of efficient aerobic oxidation of a variety of aromatic aldehydes. More importantly, they exhibited high substrate tolerance toward toxic furans. E. coli co-expressing vanillin dehydrogenase and NOX (E. coli_CtVDH1_NOX) enabled efficient oxidation of 250 mM of 5-hydroxymethylfurfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), providing a productivity of 3.7 g/L h. With E. coli_CtVDH2_NOX as catalyst, up to 240 mM of furfural and 5-methoxymethylfurfural (MMF) could be smoothly oxidized. 2-Furoic acid (FCA, 227 mM) and 5-methoxymethyl-2-furancarboxylic acid (MMFCA, 287 mM) were produced in fed-batch synthesis, providing the productivities of 2.0 and 5.6 g/L h, respectively.

Full text of DOI:10.1002/cctc.202000259

Accepted Article
Title: Furan carboxylic acids production with high productivity by
cofactor-engineered whole-cell biocatalysts
Authors: Xue-Ying Zhang, Xin Wang, Nan-Wei Li, Ze-Wang Guo, Min-
Hua Zong, and Ning Li
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Furan carboxylic acids production with high productivity
by cofactor-engineered whole-cell biocatalysts
Dr. Xue-Ying Zhang,a,c Xin Wang,a Dr. Nan-Wei Li,b Dr. Ze-Wang Guo,a Prof. Dr.
Min-Hua Zong,a Prof. Dr. Ning Lia,*
a School of Food Science and Engineering, South China University of Technology,
381 Wushan Road, Guangzhou 510640, China
b College of Light Industry and Food Technology, Zhongkai University of Agriculture
and Engineering, 501 Zhongkai Road, Guangzhou 510225, China
c College of Food Science and Engineering, Hainan University, 58 Renmin Road,
Haikou 570228, China
* Corresponding author.
Dr. N. Li, Email: lining@scut.edu.cn
1
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Abstract: Furan carboxylic acids are useful chemicals in various industries. In this
work, biocatalytic production of furan carboxylic acids was reported with high
productivities by cofactor-engineered Escherichia coli cells. NADH oxidase (NOX)
was introduced into E. coli harboring aldehyde dehydrogenases (ALDHs) to promote
intracellular NAD+ regeneration, thus significantly enhancing ALDH-catalyzed
oxidation. These engineered biocatalysts were capable of efficient aerobic oxidation of
a variety of aromatic aldehydes. More importantly, they exhibited high substrate
tolerance toward toxic furans. E. coli co-expressing vanillin dehydrogenase and NOX
(E. coli_CtVDH1_NOX) enabled efficient oxidation of 250 mM of 5-
hydroxymethylfurfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA),
providing a productivity of 3.7 g/L h. With E. coli_CtVDH2_NOX as catalyst, up to
240 mM of furfural and 5-methoxymethylfurfural (MMF) could be smoothly oxidized.
2-Furoic acid (FCA, 227 mM) and 5-methoxymethyl-2-furancarboxylic acid (MMFCA,
287 mM) were produced in fed-batch synthesis, providing the productivities of 2.0 and
5.6 g/L h, respectively.
2
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Introduction
In the last decades, the production of chemicals and fuels from biomass has
attracted growing interest, due to increased concerns on environmental issues (e.g.,
global warming).[1] Biobased furans such as 5-hydroxymethylfurfural (HMF) and
furfural are recognized as top-value biobased platform chemicals,[2] which are
synthesized via carbohydrate dehydration. 5-Methoxymethylfurfural (MMF), a
structural analog of HMF, is produced in the presence of methanol via carbohydrate
dehydration.[3] These furans can be upgraded to value-added chemicals via typical
chemical transformations such as hydrogenation, oxidation, esterification, and
amination.[4] For example, 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), one of
oxidation derivatives of HMF, is a versatile building block for the synthesis of
polyesters[5] and terephthalic acid (TPA)[6] as well as an interleukin inhibitor.[7] It also
shows antitumor activity.[8] 2-Furoic acid (2-furancarboxylic acid, FCA) derived from
furfural has found wide applications in pharmaceutical, agrochemical, flavor and
fragrance industries.[9] Also, 5-methoxymethyl-2-furancarboxylic acid (MMFCA) is a
starting material for the synthesis of renewable TPA[6] and 2,5-furandicarboxylic acid
(FDCA)[10], both of which were valuable monomers in the fields of polyesters,
polyamides and polyurethanes.[11]
In terms of environmental friendliness, mild degree of reaction conditions, and
selectivity, biocatalysis appears to be advantageous over chemical counterpart.[12]
Especially, biotransformations that usually proceed under mild conditions may be
preferred for the valorization of inherently unstable biobased furans.[13] Indeed,
biocatalytic upgrading of biobased furans has been of great interest.[14] Many whole
cells[15] as well as isolated enzymes[16] were exploited for selective oxidation of these
furans to furan carboxylic acids. Although some proof-of-concept studies were
previously reported,[16b, 17] biotransformation of biobased furans remains a fundamental
challenge, because these furans displayed significant toxicity and inhibition toward
biocatalysts.[18] The unsatisfactory substrate tolerance and low productivity of
biocatalysts reported previously may be the major barriers for their large-scale
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applications. Previously, our group presented a proof-of-concept study on biocatalytic
selective oxidation of HMF to HMFCA using Comamonas testosteroni SC1588
cells.[15a] Strikingly, Zhang and co-workers recently reported a high HMF-tolerant strain
(up to 300 mM) Deinococcus wulumuqiensis R12 for the oxidation of HMF to HMFCA;
the product of up to 511 mM was synthesized within 20 h in a fed-batch process, leading
to a volumetric productivity of 3.6 g/Lh.[19] Pyo and co-workers described the fed-batch
synthesis of HMFCA using Gluconobacter oxydans DSM 50049 cells, with a
volumetric productivity of approximately 2 g/L h.[15e] Zhou et al. found that G. oxydans
ATCC 621H enabled efficient synthesis of FCA from furfural that is more toxic toward
whole-cell biocatalysts than HMF, with a volumetric productivity of 1.6 g/L h.[20]
Recently, our group reported whole-cell catalytic synthesis of HMFCA and MMFCA
using Escherichia coli overexpressing aldehyde dehydrogenases (E. coli_ALDHs),
with the volumetric productivities of 2 and 1.2 g/L h, respectively.[15c, 18]
ALDHs have recently proven to be useful catalysts in synthetic chemistry by us
and others,[15c, 15d, 18, 21] which enable the oxidation of aldehydes to carboxylic acids
with oxidized nicotinamide cofactors (NAD(P)+). Aldehyde bind to the ALDH-
NAD(P)+ complex to form the thiohemiacetal, followed by oxidation to an acyl
intermediate; finally, the deacylation occurs, thus releasing carboxylic acid.[22] We
found that, in addition to furan carboxylic acids, minor furan alcohols (10-20%) were
formed as byproducts at the initial stage in the oxidation of furan aldehydes by E.
coli_ALDHs, and then these byproducts were slowly re-oxidized into acids, thus
markedly reducing the transformation efficiencies.[15c, 21a] The reason for the formation
of minor byproducts might be that NADH produced in ALDH-catalyzed oxidation
could not be efficiently oxidized to NAD+ in vivo, and thus it was utilized by unspecific
dehydrogenases present in host cells to reduce aldehydes. Therefore, we envisioned that
the byproduct formation would be significantly alleviated by a cofactor engineering
strategy, which is capable of effectively promoting the conversion of intracellular
NADH to NAD+.[23] NADH oxidase (NOX) is able to oxidize NADH to NAD+ with O2,
producing H2O or H2O2 as byproduct.[24] In this work, therefore, we engineered
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intracellular redox environment of E. coli_ALDHs through the introduction NOX from
Lactobacillus brevis into these biocatalysts (Scheme 1), thus greatly intensifying
catalytic oxidation of aldehydes. The substrate scope of these cofactor-engineered
biocatalysts and their tolerance toward furans were examined. Additionally, the fed-
batch synthesis of three valuable furan carboxylic acids including HMFCA, FCA and
MMFCA was performed to achieve high productivities.
Scheme 1. Oxidation of aldehydes to carboxylic acids by cofactor-engineered whole-
cell biocatalysts
Materials and Methods
Chemical and biological materials
Protein marker, restriction endonucleases, T4 DNA ligase, and MMFCA (99%) were
purchased from Thermo Fisher Scientific GmbH (Schwerte, Germany). LA Taq Hot
Start polymerase was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).
The reagent kits for constructing recombinant plasmids were purchased from Generay
Biotech Co., Ltd (Shanghai, China). Isopropyl β-D-1-thiogalactopyranoside (IPTG),
DNA marker, and ampicillin were purchased from Sangon Biotech (Shanghai, China).
FDCA (97%), and 5-formyl-2-furancarboxylic acid (FFCA, 98%) were purchased from
J&K Scientific Ltd. (Guangzhou, China). HMFCA (98%), and MMF (97%) were
bought from Adamas Reagent Ltd. (Shanghai, China). Furfural (99%), and 2,5-
bis(hydroxymethyl)furan (BHMF, 98%) were obtained from Macklin Biochemical Co.,
Ltd. (Shanghai, China). 2,5-Diformylfuran (DFF, 98%), 5-methylfurfural (97%),
furfuryl alcohol (98%), and FCA(98%) were purchased from TCI (Japan). HMF (98%)
was purchased from Aladdin (Shanghai, China). 5-Methyl-2-furoic acid (97%) was
obtained from Sigma-Aldrich (USA). The sources and purity of other chemicals are
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available as Supporting Information. The strains, plasmids, and primers used in this
study are presented in Tables S1 and S2, respectively. C. testosteroni SC1588 was
maintained in our laboratory.[15a]
Construction and cultivation of recombinant biocatalysts
The ALDH genes Ctcaldh2 (GenBank Accession No. MN460794; encoding coniferyl
aldehyde dehydrogenase 2, CtCALDH2), Ctvdh1 (GenBank Accession No. MN460795;
encoding vanillin dehydrogenase 1, CtVDH1), Ctvdh2 (GenBank Accession No.
MN4607946; encoding vanillin dehydrogenase 2, CtVDH2) and Ctsapdh (GenBank
Accession
No.
MN460792;
encoding
3-succinoylsemialdehyde-pyridine
dehydrogenase, CtSAPDH) were amplified with genomic DNA of C. testosteroni
SC1588 as template. Figure S1 shows the schematic diagrams of the construction of
recombinant expression plasmids. Briefly, the PCR products were double-digested
using the restriction enzymes BamH I and Hind III; then, the resulting products were
inserted into the expression plasmid pETDuet-1, thus generating the recombinant
plasmids pETDuet_Ctaldh. The nox gene (GenBank Accession No. ARW22543;
encoding NOX) from L. brevis was amplified with pET28a_nox as template. The
purified double-digestion fragment was inserted into the plasmids pETDuet-1 and
pETDuet_Ctaldh, thus producing pETDuet_nox and pETDuet_Ctaldh_nox,
respectively. Finally, the obtained plasmids were transformed into E. coli BL21 (DE3),
affording recombinant whole-cell biocatalysts.
Recombinant E. coli strains were pre-cultivated overnight in 30 mL of Luria-Bertani
(LB) medium containing 100 mg/L ampicillin at 37 °C and 180 r/min. Then, 100 mL
LB medium containing 100 mg/L ampicillin was inoculated with 1 mL of the overnight
culture, followed by cultivation at 37 °C and 180 r/min. When the optical density at 600
nm (OD600) reached 0.6-0.8, IPTG (as inducer) was added to the medium at a final
concentration of 0.1 mM, followed by induction at 20 °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.
Determination of intracellular NAD(H) concentrations
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The intracellular concentrations of NAD+ and NADH were determined by the reagent
kit (Keming Bio-Tech Co., China), according to the manual.
Enzyme assay
For assaying ALDH activity, 11 g/L (cell dry weight, CDW) of cells were added into 4
mL of K2HPO4-NaH2PO4 buffer (200 mM, pH 7) containing 100 mM HMF, and
incubated at 30 °C and 150 r/min for 10 min. The formation of HMFCA was monitored
by HPLC. One unit of enzyme activity (U) was defined as the amount of the cells (CDW)
which produce 1 μmol HMFCA per minute under the above conditions.
The NOX activity was spectrophotometrically determined at 30 °C by monitoring the
NADH oxidation at 340 nm.[25] The reaction mixture contains 0.2 mM NADH, an
appropriate amount of crude enzyme (the supernatant of cell lysate), and K2HPO4-
NaH2PO4 buffer (50 mM, pH 7). One unit of enzyme activity (U) was defined as the
amount of enzyme that catalyzes the oxidation of 1 μmol NADH per minute under the
above conditions.
General procedure for biocatalytic oxidation of aldehydes
Typically, 4 mL of K2HPO4-NaH2PO4 buffer (200 mM, pH 7) containing 100-280 mM
of aldehydes and 4-22 g/L (CDW) of microbial cells was incubated at 30 °C and 150
r/min. Aliquots were withdrawn from the reaction mixtures 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). All the
experiments were conducted at least in duplicate, and the values were expressed as the
means ± standard deviations.
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 (Waters, USA). The
detailed methods and HPLC spectra are available as Supporting Information.
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Results and Discussion
Engineering whole-cell biocatalysts for HMF oxidation
With CtVDH1 as a model dehydrogenase, NOX was co-expressed with this
enzyme in E. coli BL21 (DE3) for promoting NAD+ recycling (Figure S1). E. coli
without NOX was used as the control. Based on SDS-PAGE analysis (Figure S2),
soluble expression of these heterologous proteins was implemented in E. coli. These E.
coli strains were exploited for the oxidation of HMF 1 (Figure 1a). As expected, the
production of byproduct BHMF was significantly reduced with cofactor-engineered E.
coli (E. coli_CtVDH1_NOX) as catalyst compared to that with E. coli_CtVDH1 (3%
vs 12%, Figure 1b). More interestingly, the synthesis of the desired product HMFCA
was considerably enhanced with the engineered cells; HMFCA was obtained with a 96%
yield within 2 h (Figure 1b). It is in good agreement with previous reports.[23a, 26] To
explain the obtained results, intracellular concentrations of NAD+ and NADH were
determined (Figure 1c). It was interestingly found that the concentration ratios of NAD+
to NADH ([NAD+]/[NADH]) reached 3.4 in E. coli_ CtVDH1_NOX, which is 2-fold
higher than that (approximately 1.7) in the control. Higher intracellular
[NAD+]/[NADH] might not only significantly promote biocatalytic HMF oxidation, but
also alleviate the HMF reduction, likely due to functional expression of NOX. To verify
the assumption, the NOX activities of the cell supernatants as well as the ALDH
activities of the recombinant cells were assayed (Figure 1d). As shown in Figure 1d, the
cells without the incorporation of CtVDH1 displayed extremely low ALDH activities,
whereas high ALDH activities (112-129 U/g) were observed with the strains expressing
CtVDH1. More importantly, the cell supernatants of cofactor-engineered E. coli
displayed high NOX activities (14-16 U/mg). In contract, almost no NOX activities
were found in the strains without the introduction of NOX. Therefore, improved
synthesis of HMFCA with E. coli_CtVDH1_NOX might be attributed to functional
expression of NOX capable of efficiently recycling NAD+ from NADH.
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Figure 1. Biocatalytic oxidation of HMF 1 by recombinant cells (a), time courses of the oxidation
reaction (b), intracellular concentration ratio of NAD+ to NADH (c), and ALDH and NOX activities
(d). Symbols in Figure 1b: E. coli_CtVDH1 (solid symbols), E. coli_CtVDH1_NOX (open
symbols). Conditions for Figure 1b: 100 mM HMF, 11 g/L (CDW) microbial cells, 4 mL K2HPO4-
NaH2PO4 buffer (200 mM, pH 7), 30 °C, 150 r/min; conditions for Figure 1c and d are available in
experimental section.
Encouraged by the above results, other biocatalysts including E. coli_CtVDH2, E.
coli_CtCALDH2, and E. coli_CtSAPDH were cofactor-engineered by introducing
NOX (Figure S3). Also, functional expression of these proteins was readily achieved
(Table S3). In the cases of E. coli_CtCALDH2 and E. coli_CtSAPDH, the introduction
of NOX caused the reduced ALDH activities of the recombinant cells (Table S3, entries
5 and 8). For E. coli_CtCALDH2_NOX, a comparable ALDH activity was retained
when its induction temperature was lowered to 16 °C (Table S3, entry 6). However, this
strategy was ineffective for E. coli_CtSAPDH_NOX (Table S3, entry 9). Then, the
catalytic performances of these engineered strains with good ALDH activities were
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evaluated for the oxidation of HMF (Table 1). Both E. coli_CtVDH1_NOX and E.
coli_CtVDH2_NOX provided the improved results in the HMF oxidation compared to
their counterparts without cofactor engineering (Table S4). In addition, most engineered
biocatalysts showed good catalytic activities and excellent substrate tolerance. Both E.
coli_CtVDH1_NOX and E. coli_CtVDH2_NOX enabled efficient oxidation of up to
250 mM of HMF to HMFCA, with the yields of more than 92% (Table 1, entries 2 and
5). Compared to E. coli_CtVDH1 recently reported by us,[15c] E. coli_CtVDH1_NOX
displayed higher substrate tolerance (200 vs 250 mM) and selectivity (92% vs 95%) in
the batch process as well as a higher volumetric productivity (2.2 vs 3.7 g/L h).
Table 1. Whole-cell catalytic synthesis of HMFCA from HMF 1
Entry Catalyst
HMF conc. Time HMF conv. HMFCA
(mM)
200
250
100
200
250
100
100
(h)
7
(%)
yield (%)
97 ± 2
95 ± 2
94 ± 2
92 ± 1
92 ± 1
89 ± 2
87 ± 2
1
2
3
4
5
6
7
E. coli_CtVDH1_NOX
>99
>99
>99
>99
>99
92 ± 1
>99
E. coli_CtVDH1_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtCALDH2_NOX a
E. coli_CtSAPDH
9
2
9
12
6
2
Reaction conditions: 100-250 mM HMF 1, 11 g/L (CDW) microbial cells, 4 mL K2HPO4-NaH2PO4
buffer (200 mM, pH 7), 30 °C, 150 r/min; tuning pH to 7 every 2 h using NaHCO3.
a: Protein expression was induced at 16 ºC during cell cultivation.
Elucidation of substrate scope
To broaden their applications in synthetic chemistry, the substrate scope of these
biocatalysts was studied, in which a group of aromatic aldehydes was tested (Figure 2).
The four biocatalysts displayed good catalytic performances toward most of substrates.
The yields of some target products were as high as >99%. Of the four biocatalysts tested,
E. coli_CtVDH2_NOX proved to be the most versatile catalyst in the oxidation of these
aromatic aldehydes; all target carboxylic acids were obtained with excellent yields
(more than 91%). With DFF 2 as substrate, it could be sequentially oxidized to FFCA
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3 and FDCA. As shown in Figure 2, DFF 2 was a good substrate of most of biocatalysts,
except for E. coli_CALDH2_NOX, since its conversions reached 99% in 12 h (data not
shown). In the oxidation of DFF 2, the FDCA yields depended on the catalytic activities
of biocatalysts toward FFCA 3. E. coli_CtVDH2_NOX showed a good activity toward
FFCA 3, and thus a high FDCA yield (96%) was obtained in the oxidation of DFF 2
with this catalyst. In contract, E. coli_CtSAPDH with a modest catalytic activity toward
FFCA 3 (leading to 63% yield of FDCA) provided a moderate FDCA yield (52%) in
the oxidation of DFF 2. Other furan derivatives including compounds 4-7 were well
accepted as substrates by these catalysts; target furan carboxylic acids were afforded
with good yields (84->99%).
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Figure 2. Substrate scope of these whole-cell biocatalysts. Reaction conditions: 20 mM substrate,
4 g/L (CDW) microbial cells, 4 mL K2HPO4-NaH2PO4 buffer (200 mM, pH 7), 30 °C, 150 r/min,
12 h. Yields of target acids were showed in blue font, and their standard deviations were less than
4%. In the oxidation of compounds 2 and 8, the yields of the products 3 and 9 were given in
parenthesis. Protein expression was induced at 16 ºC during the cultivation of E.
coli_CtCALDH2_NOX.
a: 3 h.
b: 24 h.
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Like DFF 2, terephthalaldehyde 8 was oxidized to 4-formylbenzoic acid 9, followed by
oxidation to TPA. Compared to DFF 2, its phenyl analog 8 seemed to be a preferred
substrate for these biocatalysts. The substrate conversions reached >99% in all cases,
and TPA was obtained in the yields of more than 92% with most of biocatalysts. The
same effect was also observed in compounds 3 versus 9, but not in compounds 5 versus
12. As shown in Figure 2, the yields of benzoic acids were strongly dependent on the
nature of the substituents on the phenyl ring. Benzaldehyde with a strong electron-
withdrawing group (e.g., F atom, 4-fluorobenzaldehyde 11) could be efficiently
oxidized to target acid within 12 h, while a long reaction period (24 h) was required for
complete oxidation of 4-bromobenzaldehyde 13 with a weak electron-withdrawing
atom. For E. coli_CtVDH1_NOX, particularly, the effect of the substituent
electronegativities on biocatalytic oxidation of benzaldehydes appeared to be more
evident. The benzoic acid yields decreased from 99% to 37% with the reduced
electronegativities of the substituents (F atom: 4.0; Cl: 3.0; Br: 2.8).[27] Also, 4-
hydroxymethylbenzaldehyde 14, a structural analog of HMF 1, was a good substrate of
these biocatalysts; 4-hydroxymethylbenzoic acid was furnished with good yields within
3 h (Figure 2).
Herein we provided a versatile catalytic toolbox for the oxidation of aromatic aldehydes
to target carboxylic acids. For example, E. coli_CtVDH2_NOX exhibited high
activities toward almost all substrates tested. Besides, E. coli_CtSAPDH is a preferred
catalyst for the oxidation of compounds 12-14. These whole-cell catalysts constructed
in this work could tolerate a variety of functional groups such as the primary hydroxyl
and halogen atoms, and they were capable of selectively oxidizing the formyl group(s)
present in the substrates to the carboxyl group(s).
Batch and fed-batch synthesis of FCA
FCA is an important fine chemical, which has been widely used in the
pharmaceutical, agrochemical, flavor and fragrance industries.[9] However, furfural 4
was not only strongly inhibitory but also greatly toxic toward biocatalysts.[18] Therefore,
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the substrate tolerance of biocatalysts toward furfural 4 was evaluated, and the
optimized synthesis of FCA was performed to achieve a good productivity (Table 2).
Based on their substrate scope (Figure 2), three biocatalysts including E.
coli_CtVDH1_NOX, E. coli_CtVDH2_NOX and E. coli_CtCALDH2_NOX were
used for this purpose. As shown in Table 2, entry 1, E. coli_CtVDH1_NOX gave poor
results in the oxidation of furfural 4, despite its good catalytic performances in the
oxidation of HMF 1 (Table 1). The reason may be that furfural is much more toxic and
inhibitory toward the cells as well as toward dehydrogenases than HMF,[18, 28] especially
at high concentrations, which may be closely related to its higher hydrophobicity.
Interestingly, E. coli_CtVDH2_NOX proved to be an excellent catalyst for the
oxidation of toxic furfural 4 (Table 2). FCA was obtained with the yields of 93% and
83% at the substrate concentrations of 100 and 150 mM, respectively (Table 2, entries
2 and 4). Significantly improved FCA synthesis was achieved by increasing cell
concentrations. Furfural 4 of up to 240 mM was smoothly converted in 24 h in the
presence of 22 g/L of cells, affording FCA with a 90% yield (Table 2, entry 9).
Nevertheless, poor results were obtained at the substrate concentration of 280 mM
(Table 2, entry 10). It suggest that E. coli_CtVDH2_NOX is capable of tolerating 240
mM of furfural under the present conditions, which is much higher than those of whole-
cell biocatalysts ever reported such as Acetobacter rancens IFO3297 (50 mM),[29] G.
oxydans ATCC 621H (104 mM),[20] E. coli_CtVDH1 (50 mM)[15c] and E.
coli_CtSAPDH (100 mM).[18, 21a]
Table 2. Optimized whole-cell catalytic synthesis of FCA from furfural 4
Entry
4
conc. Catalyst
Catalyst Time 4 conv. FCA
(mM)
conc.
(h)
(%)
yield
(%)
(g/L)
1
2
3
4
5
6
100
100
100
150
150
150
E. coli_CtVDH1_NOX
E. coli_CtVDH2_NOX
E. coli_CtCALDH2_NOX a
E. coli_CtVDH2_NOX
E. coli_CtCALDH2_NOX a
E. coli_CtVDH2_NOX
4
12
12
12
12
12
6
43 ± 1 35 ± 2
4
>99
>99
93 ± 2
95 ± 3
4
4
88 ± 3 83 ± 5
68 ± 0 64 ± 2
4
11
>99
95 ± 3
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7
180
240
240
280
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
11
11
22
22
9
99
93 ± 2
8
24
24
24
64 ± 0 58 ± 0
97 ± 0 90 ± 1
30 ± 0 27 ± 1
9
10
Reaction conditions: 100-280 mM furfural 4，4-22 g/L (CDW) microbial cells, 4 mL K2HPO4-
NaH2PO4 buffer (200 mM, pH 7), 30 °C, 150 r/min; tuning pH to 7 every 2 h using NaHCO3.
a: Protein expression was induced at 16 ºC during cell cultivation.
With optimized conditions in hand, high-titer FCA synthesis was conducted with
E. coli_CtVDH2_NOX, because it could not only considerably improve the volumetric
productivity of the bioprocess, but also facilitate the downstream product purification.
Considering great toxicity and inhibition caused by high concentrations of substrate, a
fed-batch strategy was applied (Figure 3). As shown in Figure 3, approximately 100
mM of furfural was rapidly transformed to FCA within 2.5 h. Besides, furfural 4 was
also quickly exhausted in 3.5 h after the first substrate feeding. Approximately 177 mM
of FCA was produced from 194 mM furfural 4, together with 16 mM furfuryl alcohol.
The FCA yield and volumetric productivity reached around 91% and 3.3 g/L h,
respectively. Nevertheless, the catalytic efficiency of E. coli_CtVDH2_NOX sharply
decreased after the second substrate feeding, possibly due to partial catalyst inactivation
caused by toxic furfural. A period of 7 h was required for complete conversion of 50
mM substrate. Overall, FCA of up to 227 mM was produced within 13 h, along with 19
mM furfuryl alcohol. Total FCA volumetric productivity was about 2.0 g/L h, which is
higher than the highest value (1.6 g/L h)[20] in biocatalytic FCA production reported
previously.
15
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10.1002/cctc.202000259
ChemCatChem
Furfural
FCA
Furfuryl alcohol
250
200
150
100
50
st
1
nd
2
0
0
2
4
6
8
10 12 14
Time / h
Figure 3. Fed-batch synthesis of FCA by E. coli_CtVDH2_NOX. Reaction conditions: 100 mM
furfural 4, 11 g/L (CDW) microbial cells, 4 mL K2HPO4-NaH2PO4 buffer (200 mM, pH 7), 30 °C,
150 r/min. Arrows show the feed of furfural (0.2-0.4 mmol) and NaHCO3 (0.4 mmol).
Batch and fed-batch synthesis of MMFCA
As described above, MMFCA is a key starting material for the synthesis of FDCA
and TPA. To date, the studies on biocatalytic synthesis of MMFCA remain few.[15b, 18,
21a] Recently, the highest MMFCA volumetric productivity of 1.2 g/L h was reported by
our group.[18] To further improve volumetric productivity, optimized synthesis of
MMFCA were carried out with cofactor-engineered biocatalysts (Table 3). According
to the results in Figure 2, E. coli_CtVDH2_NOX and E. coli_CtCALDH2_NOX were
used as catalysts. As shown in Table 3, E. coli_CtVDH2_NOX appeared to excel over
E. coli_CtCALDH2_NOX for high-titer production of MMFCA, as indicated by
comparing MMFCA yields at the substrate concentration of 150 mM (92% vs 79%,
entries 3 and 4). The substrate tolerance of E. coli_CtVDH2_NOX was greatly
improved by simply optimizing cell concentrations. MMF 7 of up to 240 mM could be
completely converted within 9 h by 11 g/L of cells, affording MMFCA with a
quantitative yield (Table 3, entry 7). Under the same conditions, E. coli_CtVDH2_NOX
displayed much higher catalytic efficiency in the oxidation of MMF 7 than that in the
oxidation of furfural 4, as evidenced by the reaction periods for completely
transforming furans of the same concentrations (e.g., 100 mM, 6 vs 12 h, Table 3, entry
16
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10.1002/cctc.202000259
ChemCatChem
1 vs Table 2, entry 2)
Table 3. Optimized whole-cell catalytic synthesis of MMFCA from MMF 7
Entry
7 conc.
Catalyst
Catalyst
Time 7 conv.
MMFCA
yield (%)
(mM)
conc.
(g/L)
4
(h)
(%)
1
2
3
4
5
6
7
8
9
100
100
150
150
200
180
240
280
280
E. coli_CtVDH2_NOX
E. coli_CtCALDH2_NOX a
E. coli_CtVDH2_NOX
E. coli_CtCALDH2_NOX a
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
E. coli_CtVDH2_NOX
6
>99
92 ± 1
90 ± 4
92 ± 3
79 ± 3
68 ± 1
>99
4
6
98 ± 3
>99
4
9
4
9
83 ± 3
70 ± 1
>99
4
24
6
11
11
11
22
9
>99
99 ± 4
46 ± 2
73 ± 2
24
24
47 ± 1
77 ± 2
Reaction conditions: 100-280 mM MMF 7，4-22 g/L (CDW) microbial cells, 4 mL K2HPO4-
NaH2PO4 buffer (200 mM, pH 7), 30 °C, 150 r/min; tuning pH to 7 every 2 h using NaHCO3.
a: Protein expression was induced at 16 ºC during cell cultivation.
Then, fed-batch synthesis of MMFCA was performed with E.
coli_CtVDH2_NOX (Figure 4). It was found that this biocatalyst maintained a high
catalytic activity within 8 h in the synthesis of MMFCA. Around 287 mM of MMFCA
was produced from 314 mM of MMF 7, giving a yield of 91%. Its volumetric
productivity was as high as 5.6 g/Lh, which is much higher than that a recent value (1.2
g/L h) reported by us with E. coli_CtSAPDH.[18] Figure S4 shows that around 1 g of
dry cells are obtained from 4.4 g of wet cells upon lyophilization. Based on this equation,
the specific productivity of MMFCA in our recent process[18] is about 0.05 g/h per g of
cells (CDW). In terms of the specific productivity, therefore, the advantage of the
present process using E. coli_CtVDH2_NOX appeared to be more significant
compared to that with E. coli_CtSAPDH (0.5 vs 0.05 g/g h).
17
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10.1002/cctc.202000259
ChemCatChem
300
250
200
150
100
50
MMF
MMFCA
0
0
2
4
6
8
Time / h
Figure 4. Fed-batch synthesis of MMFCA by E. coli_CtVDH2_NOX. Reaction conditions:
approximately 100 mM MMF 7, 11 g/L (CDW) microbial cells, 4 mL K2HPO4-NaH2PO4 buffer
(200 mM, pH 7), 30 °C, 150 r/min. Arrows show the feed of MMF (approximately 0.4 mmol) and
NaHCO3 (0.4 mmol).
Conclusions
In summary, high substrate tolerant whole-cell biocatalysts were constructed by a
cofactor engineering strategy in this work. These engineered biocatalysts showed a
broad substrate scope, and they enabled aerobic oxidation of a group of aromatic
aldehydes to target carboxylic acids with good yields. Based on these biocatalysts, a
green and sacrificial substrate-free biocatalytic oxidation process was successfully
developed for high-titer and high-productivity synthesis of furan carboxylic acids from
biobased furans. From sustainable chemistry viewpoint, this bioprocess is
advantageous over chemical counterparts, because it uses air as the oxidant, and is
totally free of toxic catalysts and solvents. The volumetric productivity up to 5.6 g/L h
was obtained in biocatalytic synthesis of furan carboxylic acids. Although improved
results such as higher substrate tolerance, increased selectivities and productivities were
achieved, unsatisfactory long-term stability of these biocatalysts may be a major
obstacle for their large-scale applications in the conversion of toxic furans.
Understanding the molecular mechanism of the biocatalyst inactivation caused by
furans may be helpful to address this key issue, which is in progress in our laboratory.
18
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10.1002/cctc.202000259
ChemCatChem
Overall, biocatalytic aerobic oxidation may be a promising route for the valorization of
biobased furans and make contribution to sustainable synthetic chemistry.
Notes
The authors declare no competing financial interest.
Supporting Information
The Supporting Information is available free of charge on website.
Strains, plasmids, and primers used in this study, SDS-PAGE analysis of proteins,
enzyme activity assay, and HPLC analytic methods and spectra.
Acknowledgements
This research was financially supported by the National Natural Science Foundation of
China (21676103), the Natural Science Foundation of Guangdong Province
(2017A030313056), and the Guangzhou Municipal Science and Technology Project
(201804010179).
Keywords: aldehyde dehydrogenases; biobased furans; biocatalysis; cofactor
engineering; oxidation
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Graphical Abstract
Cofactor-engineered E. coli cells proved to be versatile catalysts to produce furan
carboxylic acids from toxic biobased furans with high productivities. The introduction
of NADH oxidase (NOX) into E. coli harboring aldehyde dehydrogenases (ALDHs)
significantly promoted intracellular NAD+ regeneration, thus enhancing ALDH-
catalyzed aldehyde oxidation. A group of aromatic aldehydes was efficiently oxidized
to target carboxylic acids. More importantly, these engineered whole-cell biocatalysts
displayed high substrate tolerance toward toxic furans.
23
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