Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid by Escherichia coli overexpressing aldehyde dehydrogenases

Journal Pre-proof Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid by Escherichia coli overexpressing aldehyde dehydrogenases Xue-Ying Zhang, Xiao-Yang Ou, Yi-Jing Fu, Min-Hua Zong, Ning Li

PII:

S0168-1656(19)30917-4

DOI:

https://doi.org/10.1016/j.jbiotec.2019.11.007

Reference:

BIOTEC 8546

To appear in:

Journal of Biotechnology

Received Date:

2 July 2019

Revised Date:

1 November 2019

Accepted Date:

9 November 2019

Please cite this article as: Zhang X-Ying, Ou X-Yang, Fu Y-Jing, Zong M-Hua, Li N, Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid by Escherichia coli overexpressing aldehyde dehydrogenases, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.11.007

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Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid by Escherichia coli overexpressing aldehyde dehydrogenases

Xue-Ying Zhang,a Xiao-Yang Ou,a Yi-Jing Fu,b Min-Hua Zong,a Ning Lia*

School of Food Science and Engineering, South China University of Technology,

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a

381 Wushan Road, Guangzhou 510640, China b

State Key Laboratory of Pulp and Paper Engineering, School of Light Industry and

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Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China

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* Corresponding author.

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Graphical abstract

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Dr. N. Li, Tel/Fax: +86 20 2223 6669; Email: [email protected]

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Efficient synthesis of 5-hydroxymethyl-2-furancarboxylic acid

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by Escherichia coli overexpressing aldehyde dehydrogenases

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Recombinant E. coli overexpressing ALDHs proved to be promising biocatalysts for selective oxidation of bio-based furans.

Highlights 

Recombinant E. coli strains harboring ALDHs were constructed for furan oxidation. The tolerance of E. coli_CtVDH1 toward HMF is up to 200 mM.

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HMFCA was obtained in a 92% yield in 12 h from 200 mM of HMF.

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HMFCA was produced with a productivity of 1.9 g/L h in a fed-batch process.

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Other bio-based furans were oxidized into target carboxylic acids with good

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

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yields.

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Abstract: Catalytic transformation of biomass-derived furans into value-added chemicals and biofuels has received considerable interest recently. In this work,

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aldehyde dehydrogenases (ALDHs) were identified from Comamonas testosteroni

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SC1588 for the oxidation of bio-based furans into furan carboxylic acids. Of the whole-cell biocatalysts constructed, Escherichia coli expressing a vanillin

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dehydrogenase (E. coli_CtVDH1) proved to be the best for the oxidation of 5hydroxymethylfurfural (HMF). 5-Hydroxymethyl-2-furancarboxylic acid (HMFCA) was obtained in the yield of approximately 92% within 12 h using this recombinant strain when the HMF concentration was up to 200 mM. In a fed-batch process, 292 mM of HMFCA was produced within 20.5 h, thereby providing a productivity as high 2

as 2.0 g/L h. Other furan carboxylic acids were synthesized in the yields of 83-95%. Besides, the partially purified HMF was smoothly converted into HMFCA by this recombinant strain, with a 90% yield. Keywords: bio-based platform chemicals; biocatalysis; furan carboxylic acids; oxidation; whole cells

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1. Introduction

Given the depletion of fossil resources and increasing concerns about global warming in recent years, the production of biofuels and chemicals from renewable and carbon-

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neutral biomass has attracted great interest (Sheldon, 2011; Sheldon, 2018). Furans

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such as 5-hydroxymethylfurfural (HMF) and furfural can be synthesized via carbohydrate dehydration, and they are among the U.S. Department of Energy (DOE)

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“Top 10 + 4” list of bio-based platform chemicals (Bozell and Petersen, 2010). They

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contain multi active functionalities such as aromatic ring, formyl group, and primary hydroxyl, which are responsible for their high chemical reactivity. So these bio-based

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furans can be readily upgraded into a variety of valuable products through typical chemical transformations such as oxidation, reduction, and Diels-Alder reactions (van

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Putten et al., 2013). Two important furan carboxylic acids 5-hydroxymethyl-2furancarboxylic acid (HMFCA) and 2-furoic acid (2-furancarboxylic acid, FCA) are the oxidation products of HMF and furfural, respectively. HMFCA was not only a useful building block for the synthesis of polyesters (Hirai, 1984; Todea et al., 2019) and an interleukin inhibitor (Braisted et al., 2003), but also was found to have an 3

antitumor activity (Munekata and Tamura, 1981). In addition, it was one of the key precursors in the synthesis of bio-based terephthalic acid (TPA) (Li et al., 2016; Pacheco and Davis, 2014; Pacheco et al., 2015), a monomer in the synthesis of polyethylene terephthalate (PET) that is used for the large-scale production of synthetic fibers and plastic bottles. Also, FCA has wide applications in pharmaceutical, agrochemical, flavor, and fragrance industries (Mariscal et al., 2016).

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Currently, chemical catalysis is playing a central role in the HMFCA synthesis

(Rosatella et al., 2011). In particular, HMFCA was generally synthesized via catalytic

oxidation of HMF over metal catalysts (Gupta et al., 2018). Biocatalysis appears to be

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advantageous over chemical approaches for selective oxidation of inherently unstable

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furans, since the former is performed under mild reaction conditions, and is exquisitely selective, and efficient as well as being environmentally friendly (Dong et

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al., 2018; Kroutil et al., 2004). Indeed, this green technology is currently receiving

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increasing interest in the upgrade of furans. Krystof et al. reported a chemo-enzymatic approach to HMFCA, in which peracids formed in situ via lipase-catalyzed oxidation

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of fatty acid esters with H2O2 oxidized HMF (Krystof et al., 2013). Our group found that xanthine oxidase was an excellent catalyst for selective oxidation of HMF (Qin et

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al., 2015). Recently, we exploited the promiscuous catalytic activities of hemoproteins for in situ recycling of NAD(P)+, which was combined with alcohol dehydrogenases to construct a parallel cascade for selective oxidation of HMF into HMFCA (Jia et al., 2017; Jia et al., 2019). HMF was selectively oxidized into HMFCA by BaeyerVilliger monooxygenases, with the conversions of 66-85% (Kumar and Fraaije, 4

2017). In addition to enzymes, whole cells were also used for the synthesis of HMFCA. Terasawa et al. isolated Pseudomonas syringae SF4-17 from soil for the synthesis of HMFCA (Terasawa et al., 2002). A fed-batch strategy was applied for the whole-cell catalytic production of HMFCA, due to the potent inhibition of high concentrations of substrate toward microbial cells (Mitsukura et al., 2004). We recently reported a HMF-tolerant strain Comamonas testosteroni SC1588 for the

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synthesis of HMFCA (Wen et al., 2020; Zhang et al., 2017). Knaus et al. presented a preparative scale synthesis of HMFCA using recombinant cells expressing aldehyde

dehydrogenases (ALDHs) (Knaus et al., 2018). Besides, biocatalytic synthesis of FCA

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from furfural was reported by using whole cells or enzymes (Krystof et al., 2013;

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Kumar and Fraaije, 2017; Mitsukura et al., 2004; Pérez et al., 2009; Shi et al., 2019; Zhou et al., 2017). Although biocatalysis has emerged as a promising alternative to

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chemical methods for selective oxidation of furan aldehydes (Domínguez de María

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and Guajardo, 2017; Hu et al., 2018), great challenges remain for moving this green technology into successful large-scale applications, since these furans are well-known

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inhibitors of enzymes and microorganisms (Mussatto and Roberto, 2004; Palmqvist and Hahn-Hägerdal, 2000). Except for limited examples (Krystof et al., 2013; Sayed

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et al., 2019; Wen et al., 2020; Zhang et al., 2017), therefore, the substrate concentrations used are generally low in the biotransformation of furans reported previously. In addition, the biotransformation efficiencies of furans are unsatisfactory, especially at high substrate concentrations that are highly desired in the industrial production. 5

In fact, ALDHs constitute the natural catalysts for the aldehyde oxidation. Nonetheless, this kind of enzymes were scarcely used in synthetic chemistry previously (Hollmann et al., 2011), although fundamental biochemical studies were extensively conducted for unveiling their roles in cellular metabolism (Feldman and Weiner, 1972; Ho and Weiner, 2005). Their journeys from basic science to synthetic applications are starting recently (Knaus et al., 2018; Shi et al., 2019; Wu et al., 2017).

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To understand the catalytic behaviors of C. testosteroni SC1588 and significantly

improve the catalytic performances (e.g., the catalytic efficiency, substrate tolerance, and stability) of whole cells, ALDHs responsible for the HMF oxidation were

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identified from the genome of C. testosteroni SC1588, and overexpressed in

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Escherichia coli. Recently, we found that recombinant E. coli cells expressing 3succinoylsemialdehyde-pyridine dehydrogenase (SAPDH) from C. testosteroni

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SC1588 showed much higher tolerance toward furfural (up to 100 mM) than the wild

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type strain (Shi et al., 2019). Encouraged by these results, the recombinant E. coli strains harboring ALDHs were applied as the whole-cell biocatalysts for the selective

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oxidation of HMF (Scheme 1). It was interestingly found that the recombinant strains exhibited the improved substrate tolerance as well as much higher catalytic

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efficiencies as compared to the wild type strain. In addition, a higher concentration of HMFCA in the reaction mixture was produced through a fed-batch strategy. Biotransformation of other bio-based furan aldehydes was performed using the recombinant E. coli_CtVDH1. It was found that most target carboxylic acids were obtained with good yields. To reduce the cost, the partially purified HMF was tried to 6

serve as the substrate for biocatalytic synthesis of HMFCA.

Scheme 1. Selective oxidation of HMF into HMFCA

2. Materials and Methods

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2.1. Biological and chemical materials

Restriction endonucleases, T4 DNA ligase, and 5-methoxymethyl-2-furancarboxylic

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acid (MMFCA, 99%) were purchased from ThermoFisher Scientific GmbH

(Schwerte, Germany). LA Taq Hot Start polymerase was purchased from TaKaRa

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Biotechnology Co., Ltd. (Dalian, China). The kits used for constructing recombinant

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plasmids were purchased from Generay Biotech Co., Ltd (Shanghai, China). Isopropyl-β-D-thiogalactoside (IPTG), DNA marker, ampicillin, and kanamycin were

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purchased from Sangon Biotech (Shanghai, China). 5-Formyl-2-furancarboxylic acid (FFCA, 98%), and 2,5-furandicarboxylic acid (FDCA, 97%) were purchased from

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J&K Scientific Ltd. (Guangzhou, China). HMFCA (98%), and 5-

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methoxymethylfurfural (MMF, 97%) were bought from Adamas Reagent Ltd. (Shanghai, China). Furfural (99%), 2,5-bis(hydroxymethyl)furan (BHMF, 98%), and benzaldehyde (>98.5%) 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). Benzoic acid (99.5%) was obtained from 7

Damao Chemical Reagent Ltd. (Tianjin, China). Propidium iodide (PI, ≥94%) was obtained from Simga-Aldrich (USA). The gene sequences, cloning and heterologous expression of ALDHs are available as supplementary material. Recombinant E. coli strains were cultivated in Luria-Bertani (LB) medium (pH 7.2) containing 10 g/L tryptone, 5g/L yeast extract, and 10 g/L NaCl. Nutrient broth (pH 7.2) containing 10 g/L peptone, 3 g/L beef extract, and 5 g/L NaCl was used for C. testosteroni SC1588

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cultivation.

2.2. General procedure for whole-cell biocatalytic HMF oxidation

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Typically, 4 mL of phosphate buffer (200 mM, pH 7) containing 100 mM HMF and

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50 mg/mL (wet weight) recombinant E. coli cells was incubated at 30°C and 150 r/min. Aliquots were withdrawn from the reaction mixtures at specified time intervals

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and diluted with the corresponding mobile phase prior to HPLC analysis. The

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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

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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

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means ± standard deviations.

2.3. Synthesis of HMFCA by a fed-batch strategy The reaction mixture containing 8 mL phosphate buffer (200 mM, pH 7), 50 mM HMF, and 50 mg/mL (wet weight) E. coli_CtVDH1 cells was incubated at 30°C and 8

150 r/min. When HMF was almost used up, supplementation of HMF (approximately 0.4 mmol) and NaHCO3 (0.4 mmol) was repeatedly conducted. After 20.5 h, the cells were isolated from the reaction mixture by centrifugation, and added into the fresh phosphate buffer (200 mM, pH 7) containing 50 mM HMF for the second run. The changes in the concentrations of various compounds were monitored by HPLC. The initial reaction rate (V0, namely the catalytic activity of the biocatalyst) was calculated

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on the basis of the decrease in the HMF concentrations in the initial reaction stage (generally 10 min). The relative activity was defined as the ratio of the catalytic

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activity of the used biocatalyst to that of the intact cells.

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2.4. Cell viability assay

The cell viability was measured by PI uptake and flow cytometry according to a

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recent method (Shi et al., 2019), with some modifications. Briefly, the reaction

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mixtures containing the cells were sampled at the designated time and diluted to 106 colony-forming units per mL with phosphate buffer (pH 7.2). The cells were stained

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with 10 μg/mL PI for 15 min in the dark, and the cell viability was assayed using a CytoFLEX flow cytometer (Beckman Coulter Inc., USA). The fluorescence emission

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was recorded at 630 nm upon excitation at 488 nm. Data were acquired and analyzed using the CytExpert software. The cell viability was expressed as the percentage of the cells unstained with PI in the total cells.

2.5. Synthesis of HMF from fructose 9

HMF was synthesized from fructose according to a previous method (Liu et al., 2014), with some modifications. Briefly, 5 g of fructose, 0.1 g of AlCl3, 0.2 g of concentrated sulfuric acid (98%), and 0.4 g of H3PO4 (85%) were successively added into 100 mL of N, N-dimethylformamide (DMF). After incubation at 120°C for 20 min, 10 M NaOH solution was added to neutralize the reaction solution. Then DMF was removed by reduced pressure distillation at 120°C, affording brown viscous

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slurry. The brown slurry of 5 g was extracted 5 times by 20 mL of the mixture of ethyl acetate/hexane (4:3, v/v), followed by the combination of the organic phases. Upon organic solvent evaporation, the partially purified HMF was obtained. Its purity is

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approximately 70%, based on the NMR analysis (Fig. S2).

2.6. HPLC analysis

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The reaction mixtures were analyzed on a Zorbax Eclipse XDB-C18 column (4.6 mm

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× 250 mm, 5 μm, Agilent, USA) by using a reversed phase HPLC equipped with a Waters 996 photodiode array detector (Waters, USA). The mobile phase for HPLC

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analysis was a mixture of acetonitrile and 0.4% (NH4)2SO4 aqueous solution (pH 3.5, 1/9, v/v) at a flow rate of 0.6 mL/min. The retention times of HMFCA (maximum

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absorption wavelength: 252 nm), BHMF (223 nm), and HMF (283 nm) were 6.2, 8.1, and 9.8 min, respectively. The methods for analyzing other substances are available as supplementary material.

3. Results and Discussion 10

3.1. Screening whole-cell biocatalysts Based on the functional annotations of the predicted coding sequences, seven putative ALDH genes including two ALDHs (CtALDH1 and CtALDH2), two coniferyl aldehyde dehydrogenases (CtCALDH1 and CtCALDH2), two vanillin dehydrogenases (CtVDH1 and CtVDH2) and a CtSAPDH were chosen from the genome of C. testosteroni SC1588 for constructing recombinant strains. Theses

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ALDHs were cloned and heterologously expressed in E. coli BL21 (DE3) with C-

terminal six-His tag. In a preliminary study on whole-cell biocatalytic oxidation of

HMF (Fig. S3), it was found that, with the exception of the recombinant E. coli strains

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expressing CtALDH1 and CtALDH2, other recombinant strains were capable of

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efficient conversion of HMF into HMFCA in the yields of 71-96% within 3 h when the substrate concentration was 20 mM. In contrast, HMFCA was obtained in a yield

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of only 5% in the control where E. coli cells harboring plain vector worked as the

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biocatalyst. It suggests that the five ALDHs including CtCALDH1, CtCALDH2, CtVDH1, CtVDH2, and CtSAPDH present in C. testosteroni SC1588 are responsible

Conversion / Yield (%)

100

HMF

HMFCA

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80

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for the synthesis of HMFCA.

60 40 20

0

1 H H2 H1 H2 DH PD LD tVD tVD AL tSA tCA i_C i_C li_C i_C col col l o . . c o E E c E. E.

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CtC li _ . co

l ntro Co

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Fig. 1. Biocatalytic oxidation of HMF using recombinant E. coli cells. Reaction condition: 100 mM HMF, 50 mg/mL (wet weight) cells, 4 mL phosphate buffer (200 mM, pH 7), 30°C, 150 r/min, 5 h. The E. coli_pET28a (plain vector) was used as the control.

Whole cells of recombinant E. coli expressing ALDHs were used for biocatalytic

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oxidation of HMF (Fig. 1), because the purified enzymes are unstable (data not shown). In addition, a complex system for regenerating oxidized nicotinamide cofactors is required for dehydrogenase-catalyzed oxidation using the purified

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enzymes. As shown in Fig. 1, almost all recombinant strains displayed good catalytic

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activities in the oxidation of HMF when the substrate concentration was 100 mM, except for E. coli_CtCALDH1. The substrate conversions of 97-100% were achieved

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within 5 h. On the contrary, only 7% of HMF was transformed in the control. It

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suggests that ALDHs expressed in E. coli contribute to the HMF transformation. Of the whole-cell biocatalysts tested, E. coli_CtVDH1 gave the highest HMFCA yield

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(89%). In addition to HMFCA, the reduction product BHMF (approximately 11%) was also formed as the byproduct. Also, the BHMF production (8-20% yields) was

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observed when other recombinant strains were tested as the biocatalysts in this work. It might be ascribed to the catalytic behaviors of such reductases as alcohol dehydrogenases and aldehyde reductases inherently present in the host cells.

3.2. Substrate tolerance of recombinant E. coli_CtVDH1 12

100

HMFCA yield (%)

80 100 mM 150 mM 175 mM 200 mM 225 mM

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40

20

0 0

2

4

6

8

10

12

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Time (h)

Fig. 2. Effect of substrate concentrations on biocatalytic synthesis of HMFCA.

Reaction condition: HMF of the designated concentration, 50 mg/mL (wet weight) E.

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coli_CtVDH1 cells, 4 mL phosphate buffer (200 mM, pH 7), 30°C, 150 r/min. pH of the reaction mixture was adjusted to approximately 7 every 2 h by adding NaHCO3

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during the reaction.

Substrate tolerance of the biocatalysts is of great importance for their synthetic

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applications, especially in the transformation of toxic and inhibitory substances. Therefore, the substrate tolerance of E. coli_CtVDH1 was evaluated (Fig. 2).

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Noteworthy, this whole-cell biocatalyst was capable of tolerating a HMF

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concentration as high as 200 mM, which is slightly lower than the corresponding value (250 mM) of Gluconobacter oxydans cells (Sayed et al., 2019). However, the cell concentration used in the latter is much higher than that in this work (24 vs. 12 mg/mL on a dry matter basis). As shown in Fig. 2, HMF of 200 mM was completely converted within 12 h, affording HMFCA with a yield of approximately 92%. 13

However, the substrate conversion (about 18%) sharply decreased when its concentration increased to 225 mM, likely due to the significantly negative effects of HMF on the biocatalyst. Compared to the wild type strain C. testosteroni SC1588 (Zhang et al., 2017), recombinant E. coli_CtVDH1 cells showed a slightly higher substrate tolerant level (180 vs. 200 mM). Notably, the catalytic efficiencies were significantly improved with recombinant cells as the biocatalyst. The reaction period

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required for the transformation of 200 mM HMF was approximately 12 h with E.

coli_CtVDH1, while being 36 h for the transformation of 160 mM HMF with the wild type strain C. testosteroni SC1588 (Zhang et al., 2017). Recently, Knaus et al.

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reported that ALDHs from bovine lens, E. coli and Pseudomonas putida brought

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about the oxidation of a large group of aldehydes including HMF; however, significant substrate inhibition occurred when the HMF concentrations were more

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than 20 mM (Knaus et al., 2018).

3.3. Fed-batch synthesis of HMFCA st

nd

1 run

150

100

st

nd

80

Relative activity Cell viability

60

40 th

4

st

th

5

rd

1

100

HMF HMFCA BHMF

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200

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Concentration (mM)

250

2 run

3

1

th

6

nd

2

2

20

50

0

Relative activity / cell viability (%)

300

0 0

5

10

15

20

25

30

35

40

Time (h)

Fig. 3. Biocatalytic synthesis of HMFCA by a fed-batch strategy. Reaction conditions: 14

50 mM HMF, 50 mg/mL (wet weight) E. coli_CtVDH1 cells, 8 mL phosphate buffer (200 mM, pH 7), 30°C, 150 r/min. After the substrate was almost used up, HMF (approximately 0.4 mmol) and NaHCO3 (0.4 mmol) were supplemented; the wholecell biocatalyst was isolated from the reaction mixture after 20.5 h, and then added into the fresh reaction mixture for the 2nd run. The relative activity of the intact cells

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was defined as 100%. Arrows show the feed of HMF.

The achievement of a high concentration of the product in the reaction mixture is

highly desired, because it can not only significantly improve the process productivity,

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but also facilitate the product purification. Given the high toxicity and strong

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inhibition of HMF toward microbial cells, a fed-batch strategy was applied for the synthesis of HMFCA (Fig. 3). As shown in Fig. 3, the cells were capable of efficient

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transformation of HMF within 15.5 h, as evidenced by the comparable reaction

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periods (2-3 h) required for complete conversion of HMF in each batch. However, the reaction period was greatly extended to 5.5 h for the conversion of 50 mM HMF after

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the 6th substrate feeding. Previously, we have demonstrated that sodium salt of HMFCA would have a significantly inhibitory effect on the catalytic activity of C.

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testosteroni SC1588 cells when its concentrations were higher than 200 mM (Zhang et al., 2017). More than 240 mM of furan carboxylate was produced in the reaction mixture after 15.5 h (Fig. 3). So the product inhibition might partially contribute to the reduced catalytic activity after the 6th substrate feeding. On the other hand, the changes in the relative activities of the cells during the reaction were monitored (Fig. 15

3). After the reaction of 20.5 h, the cells were isolated and added into the fresh reaction mixture, in which the product inhibition was free. However, the cells only displayed the relative activity of 64% at 20.5 h, suggesting the partial inactivation of the biocatalyst. HMF was a well-known toxic substance toward the cells. Indeed, the cell viability markedly decreased from the original 95% to approximately 54% after 20.5 h (Fig. 3), which might explain the reduced catalytic activity of the cells. And

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even lower cell viability (29-39%) was observed after 23.5 h, thus resulting in the low

HMF oxidation rates. Overall, HMFCA of approximately 292 mM was produced from 333 mM HMF within 20.5 h, together with 32 mM BHMF. The yield of HMFCA was

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around 88%. The productivity of HMFCA was around 2.0 g/L h in the 1st run, which

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is comparable to the corresponding value in the synthesis of HMFCA using 24 mg G. oxydans cells (dry weight) per mL buffer (Sayed et al., 2019). In the 2nd run,

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approximately 149 mM of HMFCA was synthesized within 18 h, along with 5 mM

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BHMF.

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3.4. Synthesis of furan carboxylic acids Table 1. Oxidation of aldehydes into carboxylic acids using E. coli_CtVDH1 cells

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Entry Substrate

Product

Time (h)

Conversion Yield (%) (%)

1

Furfural

FCA

12

97 ± 1

90 ± 0

2 3 4 5

DFF FFCA MMF 5-Methylfurfural

FFCA FDCA MMFCA 5-Methyl-2-furoic acid

6 24 11 72

44 ± 1 100 96 ± 1 95 ± 1

29 ± 1 83 ± 1 95 ± 3 86 ± 2

16

6

Benzaldehyde a

Benzoic acid

12

100

91 ± 1

Reaction conditions: 50 mM aldehydes, 50 mg/mL (wet weight) cells, 4 mL phosphate buffer (200 mM, pH 7), 30°C, 150 r/min a

: addition of 6% DMSO

In addition to HMF, other furan aldehydes were examined as the substrates (Table 1).

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Furfural proved to be a good substrate, which was completely converted within 12 h. The desired product FCA was obtained with a yield of 90%, which is slightly lower

than that (96%) with recombinant E. coli harboring SAPDH as the biocatalyst (Shi et

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al., 2019). Compared to the wild type strain C. testosteroni SC1588 (Zhang et al.,

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2017), the recombinant strain showed a much higher catalytic activity in the furfural oxidation, which was clearly evidenced by the substantially reduced reaction period

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(96 h for the former vs. 12 h in Entry 1). However, the substrate conversion was poor when DFF served as the substrate, likely due to the high toxicity of this dialdehyde

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toward the cells, because substantial cell decay was observed after the reaction of 6 h. FFCA was produced as the major product in the yield of 29%, along with HMF (Entry

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2). In addition, no FDCA was detected in the reaction mixture. As shown in Table 1,

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FDCA, an attractive bio-based alternative to TPA (Zhang and Dumont, 2017), was produced from FFCA in the yield of 83% (Entry 3). MMF obtained via fructose dehydration in the presence of methanol is an analog of HMF (Alipour et al., 2017; Zhu et al., 2011), but the former is more stable than the latter. It was found that, like HMF, MMF could be quickly transformed into the corresponding acid by this 17

recombinant strain. An excellent yield (95%) was achieved (Entry 4). Nevertheless, the oxidation reaction became very slow with 5-methylfurfural as the substrate. A long reaction period (72 h) was required to achieve a high substrate conversion with this chemical as the substrate, while HMF of the same concentration was converted completely within 2 h. It indicates that the presence of 5-hydroxyl group facilitates biocatalytic oxidation of the formyl group in HMF. Also, it was verified by the fact

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that the oxidation rate of MMF in which the primary hydroxyl is blocked by the methyl is much lower than that of HMF (Entry 4). 5-Methyl-2-furoic acid was

obtained in the yield of 86% after 72 h (Entry 5). In addition to furans, benzaldehyde

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was found to be an appropriate substrate of this recombinant strain. Benzoic acid was

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afforded with a high yield (91%) within 12 h (Entry 6).

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3.5. Synthesis of HMFCA from partially purified HMF HMF

20

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60

40

HMFCA

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80

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Conversion / Yield (%)

100

0

50

100

150

200

HMF concentrations (mM)

Fig. 4. Biocatalytic synthesis of HMFCA from partially purified HMF. Reaction conditions: a designated concentration of partially purified HMF, 50 mg/mL (wet weight) cells, 4 mL phosphate buffer (200 mM, pH 7), 30°C, 150 r/min. pH was 18

adjusted to approximately 7 every 2 h by adding NaHCO3 during the reaction.

Finally, the partially purified HMF acted as the substrate for the whole-cell catalytic synthesis of HMFCA (Fig. 4), since it would considerably reduce the cost as well as the environmental impact, due to use of less chemicals and energy for the HMF purification. HMF was synthesized by us via acid-catalyzed dehydration of fructose,

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and then organic solvent extraction was conducted. The partially purified HMF was

obtained upon solvent evaporation, with the purity of approximately 70%. As shown in Fig. 4, almost all HMF was exhausted when its concentrations were less than 150

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mM. In addition, good yields of HMFCA (86-90%) were afforded, which are

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comparable to the results obtained with HMF of high purity as the substrate (Fig. 2). It suggests that it appears to be feasible to use the partially purified HMF as the

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substrate for this recombinant strain when the substrate concentrations are less than

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150 mM. However, the substrate conversion (85%) as well as the HMFCA yield (80%) slightly decreased when the substrate concentration increased to 200 mM.

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Based on 1H NMR analysis (Fig. S2), it might be attributed to the detrimental effect

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of the residual solvent DMF present in the partially purified HMF on the biocatalyst.

4. Conclusions In summary, we have successfully identified ALDHs responsible for the HMF oxidation from Comamonas testosteroni SC1588 and constructed efficient whole-cell biocatalysts for selective oxidation of bio-based furans. E. coli_CtVDH1 displayed a 19

high tolerant level toward HMF. More importantly, its catalytic efficiency was very high in the HMF oxidation. HMFCA of approximately 290 mM was produced in 20.5 h by a fed-batch strategy, and its productivity was up to 2.0 g/L h. In addition, various bio-based furans such as furfural and MMF were selectively transformed into the

Conflicts of interest The authors declare to have no competing interests

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Acknowledgements

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corresponding carboxylic acids with good yields via this biocatalytic approach.

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This research was financially supported by the National Natural Science Foundation of China (21676103), the Natural Science Foundation of Guangdong Province

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(201804010179).

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(2017A030313056), and the Science and Technology Project of Guangzhou City

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Supplementary material

Gene sequences, cloning and heterologous expression of ALDHs, 1H NMR spectrum

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of the partially purified HMF (Fig. S2), the preliminary screening of biocatalysts (Fig. S3), HPLC spectra (Fig.s S4-9).

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