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Article

Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural by Bacillus subtilis J8M8

by
Xiangling Jiang
1,2,
Changtong Chen
1,2,
Mingxia Jiao
1,2,
Jianqi He
1,2,
Xuechun Han
3,
Tengfei Wang
1,2,*,
Di Huang
1,2,
Yi Jiang
1,2,
Hongling Liu
1,2 and
Haibo Yuan
1,2,*
1
State Key Laboratory of Green Papermaking and Resource Recycling, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
2
Shandong Provincial Key Laboratory of Biosensing and Microbial Intelligent Metabolic Regulation, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
3
Luwei Pharmaceutical Group Co., Ltd., Zibo 255100, China
*
Authors to whom correspondence should be addressed.
Microorganisms 2026, 14(2), 425; https://doi.org/10.3390/microorganisms14020425
Submission received: 15 January 2026 / Revised: 8 February 2026 / Accepted: 8 February 2026 / Published: 11 February 2026
(This article belongs to the Section Microbial Biotechnology)
Browse Figures
Versions Notes

Abstract

2,5-Furandicarboxylic acid (FDCA) is an important bio-based platform compound that can be synthesized through the biotransformation of 5-hydroxymethylfurfural (HMF). However, the limited availability of safe microbial strains is a major constraint in the whole-cell catalysis of HMF to FDCA. In this study, a strain capable of catalyzing the conversion of HMF to FDCA, Bacillus subtilis J8M8, was identified. Under optimized whole-cell catalytic conditions, the wild-type strain produced 33.1 mM FDCA with a yield of 41.4%. To enhance FDCA production, HMF/furfural oxidoreductase (HmfH), PQQ-dependent alcohol dehydrogenase (ADH), and aryl-alcohol oxidase (MaAAO) were co-expressed in B. subtilis J8M8. As a result, FDCA production increased to 72.3 mM, with a yield of 90.4%. Further optimization of the engineered strain improved FDCA production to 83.3 mM and yield to 92.6%, representing a 2.52-fold increase over that of the wild-type strain. This study establishes a foundation for the safe and sustainable production of FDCA from HMF.

Graphical Abstract

1. Introduction

2,5-Furandicarboxylic acid (FDCA) is an aromatic monomer derived from biomass and has been recognized by the U.S. Department of Energy as one of the 12 most value-added chemicals [1]. 5-Hydroxymethylfurfural (HMF), which can be obtained from biomass feedstock, serves as a key intermediate linking biomass to platform compounds [2]. Various important platform compounds can be generated using HMF as substrate, due to the presence of both an aldehyde and a hydroxyl group in its structure. Among them is FDCA, which can be produced through three sequential oxidation steps [3]. FDCA is structurally and chemically similar to petroleum-based terephthalic acid and can be used to synthesize polyethylene furanoate (PEF). PEF is a potential substitute for polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) [4]. The thermal properties of PEF are superior to those of PET [5]. Its higher heat resistance and lower processing temperatures make it suitable for specific food and beverage packaging applications [6]. PEF produced from FDCA is also widely used in the electronics, electrical, automotive, medical, and construction industries [7]. Moreover, FDCA-based PEF production reduces greenhouse gas emissions by approximately 50% compared to that of petroleum-based plastic production, highlighting its potential for promoting sustainability and environmental protection [8,9,10,11].
Chemical catalysis is the most widely used method for synthesizing FDCA from HMF [12]. However, the oxidants employed in chemical synthesis—such as KMnO4, N2O4, and HNO3—can negatively affect the environment [13]. Additionally, the use of noble metal catalysts is often expensive and significantly increases the production cost of FDCA [14]. In recent years, biosynthetic approaches have emerged as more environmentally friendly alternatives due to their high catalytic selectivity and the ability to operate under milder reaction conditions, thereby reducing the formation of harmful byproducts [15]. Whole-cell catalysis operates under mild conditions, does not require additional cofactors, and enables the integration of multistep catalytic reactions within a single cell. As a result, it has garnered increasing attention from researchers [16].
Various microorganisms capable of producing FDCA from HMF have been identified recently. Burkholderia cepacia H-2 and Methylobacterium radiotolerans G-2, which catalyze the oxidation of HMF to FDCA, were isolated from soil, but the FDCA yield was only approximately 50% [17,18]. Mycobacterium sp. MS1601 oxidizes HMF to FDCA with a yield of 60% at an HMF concentration of 10 mM [19]. Klebsiella oxytoca NCIM 2694 converted 98% of HMF into FDCA with 58% selectivity after 96 h [20]. Cupriavidus basilensis HMF14 effectively degrades furfural and HMF. After analyzing its fermentation characteristics and identifying key genes involved in HMF and furfural catalysis in C. basilensis HMF14, researchers proposed a biological method for producing FDCA using whole-cell biotransformation through heterologous expression of HMF/furfural oxidoreductase (HmfH) in Pseudomonas putida S12, which significantly improved FDCA production [21]. Dijkman et al. screened for homologous genes encoding enzymes with similar metabolic roles and identified the first HMF oxidase (HMFO) from Methylovorus sp. strain MP688 [22]. Raoultella ornithinolytica BF60 was isolated from the soil, and FDCA degradation was avoided by knocking down the dcaD gene. Furthermore, HmfH and HMFO were overexpressed by optimizing gene expression elements, and genes responsible for the reduction of HMF to 2,5-bis(hydroxymethyl)furan were deleted. As a result, FDCA production reached 264.7 mM, with a yield of 96.2% [23,24,25]. In addition to single-strain whole-cell catalysis, some researchers have employed dual-strain systems. Zhang et al. combined recombinant Escherichia coli and Desmospora wulumuqiensis R12 under optimized reaction conditions, converting 180 mM HMF to FDCA with a 99% yield [26]. However, the field of whole-cell catalytic synthesis of FDCA from HMF remains in its early stages. Few strains are capable of independently synthesizing FDCA from HMF, and many are conditionally pathogenic, which limits the application of whole-cell catalysis for FDCA production [2].
In this study, a safe strain, Bacillus subtilis J8M8, was isolated from wastewater, and its catalytic performance in HMF conversion was investigated. HmfH, PQQ-dependent alcohol dehydrogenase (ADH), and aryl-alcohol oxidase (MaAAO) were co-expressed in B. subtilis J8M8 to enhance FDCA production via whole-cell catalysis. This study provides a reference for the safe biosynthesis of FDCA and lays the groundwork for the future industrialization of furan derivatives.

2. Materials and Methods

2.1. Strains, Plasmids, Chemicals, and Medium

The strains and plasmids used in this study are listed in Table 1 and expression plasmid profile are shown in Figure S1. A DNA gel extraction kit and a plasmid miniprep extraction kit were purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). A one-step cloning kit was obtained from Vazyme Biotech Co., Ltd. (Nanjing, China). A biochemical identification kit for B. subtilis was purchased from Beijing North Han Culture Collection Biotechnology Co., Ltd. (Beijing, China). Blood agar plates (supplemented with 5% sterile sheep blood) were purchased from Qingdao Hope Bio-technology Co., Ltd. (Qingdao, China). HMF, 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 5-formyl-2-furancarboxylic acid (FFCA), and FDCA were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and obtained from commercial sources. The culture medium used is as follows: luria–bertani (LB; tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L), terrific broth (TB; yeast extract 24 g/L, tryptone 12 g/L, glycerol 4 mL/L, KH2PO4 2.31 g/L, K2HPO4·3H2O 16.43 g/L) and egg yolk agar medium (tryptone 15 g/L, Beef extract powder 3 g/L, NaCl 5 g/L, Agar 15 g/L, Glucose 10 g/L, 50% Egg yolk emulsion 100 mL/L).

2.2. Isolation and Molecular Identification of Microbes That Produce FDCA from HMF

The FDCA-producing strain was isolated from the papermaking wastewater of Shandong Aosen Paper Industry Co., Ltd. (39°54′39.33″ N, 116°24′48.1824″ E; Zibo, China). A 100 µL aliquot of the wastewater was placed into a centrifuge tube and diluted 10-fold. Then, 200 µL of the diluted wastewater was spread on LB agar plates containing 20 mM HMF and incubated at 30 °C. Rapidly growing single colonies were selected, inoculated into LB broth containing 20 mM HMF, and incubated at 30 °C with shaking at 200 rpm for 24 h. The cultures were processed, and their FDCA production capacity was verified using high-performance liquid chromatography (HPLC). The strain exhibiting larger colonies and higher FDCA production was selected as the target strain.
The genomic DNA of the target strain was extracted according to the instructions of the genome extraction kit (No: DP302-02, TIANGEN Biotech Co., Ltd. (Beijing, China)). The 16S rRNA sequence of the target strain was amplified via PCR using the 27F and 1492R primers (Table S1). The PCR products were sequenced and compared with those in the National Center for Biotechnology Information (NCBI) GenBank database using Basic Local Alignment Search Tool (BLAST 2.15.0) (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 3 June 2024). MEGA 7.0 was utilized to construct phylogenetic trees using the neighbor-joining method, and the bootstrap method was used to calculate branch node support values based on 1000 replicates.
Physiological and biochemical experiments on B. subtilis J8M8 were carried out in accordance with the reagent kit guidelines (No:362442, Beijing North Han Culture Collection Biotechnology Co., Ltd., Beijing, China), employing Bacillus cereus as the positive control. In the lecithinase assay, the tested strains were inoculated onto egg yolk agar plates and incubated at 37 °C for 20 h to observe opalescent precipitation halos around the colonies. For the hemolysis assay, strains were streaked onto blood agar plates with 5% sterile sheep blood and incubated at 37 °C for 20 h. Hemolytic patterns were classified as α-hemolysis, β-hemolysis, or γ-hemolysis based on the appearance of clearance zones around the colonies.

2.3. DNA Manipulation

The amino acid and nucleotide sequences of HmfH, ADH, and MaAAO are provided in Table S2. The primers used to construct the plasmids are listed in Table S1. To construct the pSTOP-hmfH, pSTOP-adh, and pSTOP-MaAAO plasmids, the primers HmfH-F/R, ADH-F/R, and MaAAO-F/R (including 20 bp homologous sequences) were used to amplify the genes encoding HmfH, ADH, and MaAAO, respectively. The linear pSTOP plasmid was amplified using pSTOP-F/R as primers, and genes encoding HmfH, ADH, and MaAAO were cloned into the pSTOP plasmid using a one-step cloning kit based on homologous recombination. To construct the pSTOP-hmfH-adh plasmid, the genes encoding HmfH and ADH were amplified using primers HmfH(HA)-F/R and ADH(HA)-F/R, respectively. The HmfH and ADH gene fragments were used as templates for fusion PCR. The fusion PCR protocol was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 90 s, for 10 cycles, followed by storage at 4 °C. Subsequently, PCR amplification was performed using 10 µL of the fusion PCR products as the template and HmfH(HA)-F and ADH(HA)-R as primers to obtain the fusion fragment HmfH-ADH. This fragment was cloned into the pSTOP plasmid using the one-step cloning kit. To construct the pSTOP-hmfH-MaAAO plasmid, the genes encoding HmfH and MaAAO were amplified using HmfH(HM)-F/R and MaAAO(HM)-F/R as primers, respectively. Fusion PCR was then performed using HmfH(HM)-F and MaAAO(HM)-R as primers to obtain the fusion fragment HmfH-MaAAO under the same conditions as described above. The resulting HmfH-MaAAO fragment was cloned into the pSTOP plasmid using the one-step cloning kit. To construct the pSTOP-adh-MaAAO plasmid, the genes encoding ADH and MaAAO were amplified using ADH(AM)-F/R and MaAAO(AM)-F/R as primers, respectively. Fusion PCR was performed using ADH(AM)-F and MaAAO(AM)-R as primers to obtain the fusion fragment ADH-MaAAO under the same PCR conditions as above. The ADH-MaAAO fragment was cloned into the pSTOP plasmid using the one-step cloning kit. To construct the pSTOP-hmfH-adh-MaAAO plasmid, the plasmid containing the genes encoding HmfH and ADH was linearized using HA(HAM)-F/R as the primers, and the gene encoding MaAAO was amplified using MaAAO(HAM)-F/R as the primers. The linearized plasmid was then recombined with the MaAAO fragment to generate the pSTOP-hmfH-adh-MaAAO plasmid.
The constructed recombinant plasmids pSTOP-hmfH, pSTOP-adh, pSTOP-MaAAO, pSTOP-hmfH-adh, pSTOP-hmfH-MaAAO, pSTOP-adh-MaAAO, and pSTOP-hmfH-adh-MaAAO were transformed into E. coli DH5α using the CaCl2 method [27]. The recombinant plasmids were confirmed using Ver-F and Ver-R primers, followed by DNA sequencing conducted by Tsingke Biotechnology Co., Ltd. (Beijing, China). Subsequently, the correctly recombined plasmid extracted from E. coli DH5α was transformed into the competent cells of B. subtilis J8M8 via electroporation at 2000 V for 5 ms. After a 3 h resuscitation in 1 mL of antibiotic-free LB medium, a 100 μL aliquot of the cell suspension was spread onto LB agar plates supplemented with 50 μg/mL of kanamycin, followed by incubation at 37 °C for 12 h. Subsequently, a single colony was selected and identified as the recombinant B. subtilis J8M8 [28].

2.4. Optimization of Whole-Cell Catalytic Conditions of the B. subtilis J8M8

To prepare the whole-cell catalyst, B. subtilis J8M8 was streaked onto LB agar plates. Subsequently, single colonies were selected and inoculated into liquid LB medium to obtain a seed culture. This culture was then transferred (1%, v/v) into a 2 L flask containing 400 mL of TB medium and incubated at 37 °C with shaking at 220 rpm. After 24 h, the cells were harvested through centrifugation (8000× g, 4 °C, 20 min), washed twice with phosphate buffer, and finally resuspended in 10 mL aliquots in 100 mL flasks.
To investigate the effect of cell concentration on catalytic efficiency, cell concentrations (OD600) of 50, 60, 70, 80, 90, 100, and 110 were tested under reaction conditions of 100 mM HMF at 30 °C and pH 7.0 for 84 h. To assess the effect of HMF concentration on the catalytic efficiency, final HMF concentrations of 20, 40, 60, 80, 100, 120, and 140 mM were tested using a cell concentration of OD600 = 90 at 30 °C and pH 7.0 for 84 h. To evaluate the effect of pH on the catalytic efficiency, cells were resuspended in phosphate (pH 6.0, 6.5, 7.0, 7.5, and 8.0) and Tris-HCl (pH 8.0, 8.5, and 9.0) buffers, using a cell concentration of OD600 = 90, and the reaction was carried out at 30 °C for 84 h under 80 mM HMF. To examine the effect of temperature on the catalytic efficiency, the reaction temperature was maintained at 20, 25, 30, 35, 40, and 45 °C, with 80 mM HMF and a cell concentration of OD600 = 90 for 84 h.

2.5. Optimization of Whole Cell Catalytic Conditions of the BS-HAM Strain

To prepare the whole-cell catalyst, BS-HAM strain was streaked onto LB agar plates, and single colonies were selected and inoculated into liquid LB medium supplemented with 50 μg/mL of kanamycin to obtain a seed culture. This culture was then transferred (1%, v/v) into a 2 L flask containing 400 mL of TB medium supplemented with 50 μg/mL of kanamycin and incubated at 37 °C with shaking at 220 rpm. When the cells reached an OD600 of 1.2, xylose was added at a final concentration of 6 g/L to induce protein expression. After 24 h, the cells were harvested via centrifugation (8000× g, 4 °C, 20 min), washed twice with phosphate buffer, and were resuspended in 10 mL aliquots in 100 mL flasks.
To investigate the effect of cell concentration on catalytic efficiency, cell concentrations (OD600) of 70, 80, 90, 100, and 110 were tested, and the reaction was carried out with 80 mM HMF at 35 °C and pH 7.5 for 84 h. To assess the effect of HMF concentration on the catalytic efficiency, HMF was tested at final concentrations of 70, 80, 90, 100, and 110 mM, and the reaction was carried out with a cell concentration of OD600 = 90 at 35 °C and pH 7.5 for 84 h. To evaluate the effect of pH on the catalytic efficiency, cells were resuspended in phosphate (pH 6.0, 6.5, 7.0, 7.5, and 8.0) and Tris-HCl (pH 8.0, 8.5, and 9.0) buffers, using a cell concentration of OD600 = 90, and the reaction was carried out at 35 °C for 84 h with 90 mM HMF. To examine the effect of temperature on the catalytic efficiency, the reaction temperature was maintained at 20, 25, 30, 35, 40, and 45 °C, with 90 mM HMF and a cell concentration of OD600 = 90 for 84 h.

2.6. Analytical Methods

HMF, HMFCA, FFCA, and FDCA were analyzed using an HPLC LC-20A system (Shimadzu, Kyoto, Japan) equipped with a VWD detector and an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA). The mobile phase consisted of 10 mM H2SO4, with a flow rate of 0.6 mL/min. The column temperature and detection wavelength were set at 60 °C and 268 nm, respectively.

2.7. Statistical Analysis

Data are presented as mean ± Standard Deviation (SD), and all experiments were performed in triplicate. Statistical comparisons were conducted using one-way analysis of variance. Statistical analysis was performed using the Origin 9.4 software platform (OriginLab Corporation, Northampton, MA, USA).

2.8. SDS-PAGE Analysis

SDS-PAGE analysis was conducted by harvesting cultured cells through centrifugation, resuspending them in potassium phosphate buffer to an OD600 of 10, disrupting the cell suspension via ultrasonication, and subsequently centrifuging it at 12,000× g for 5 min. A 40 μL aliquot of the resulting supernatant was mixed with 10 μL of 5× protein loading buffer, heated at 99 °C for 10 min, and then 10 μL of the prepared sample was loaded onto a 4–20% precast polyacrylamide gel for electrophoresis at 130 V for 1 h. The gel was stained with 0.1% Coomassie Brilliant Blue R-250 for 30 min, followed by destaining before observation.

3. Results and Discussion

3.1. Screening and Identification of FDCA-Producing Strain

Three microorganisms capable of growing rapidly in the presence of 20 mM HMF were isolated from papermaking wastewater. The three strains (J8M8, R1 and R2) were subsequently evaluated for their ability to produce FDCA. Among them, strain J8M8 exhibited the highest FDCA production, reaching 10 mM. The R1 strain produced 4.8 mM of FDCA, whereas the R2 strain produced 1.8 mM of FDCA. Therefore, the J8M8 strain was chosen as the target strain (Figure S2). Based on the analysis of intermediates in the catalytic process, we hypothesized that the metabolic pathway of HMF to FDCA in the J8M8 strain proceeded as follows: HMF was first oxidized to HMFCA, which was subsequently oxidized to FFCA, and finally converted to FDCA through further oxidation (Figure S3, Route A).
Colonies of J8M8 strain were typically round in shape, with a rough surface, predominantly white in color, and relatively dry in texture (Figure S4). Gram staining revealed that J8M8 was a Gram-positive bacterium. The 16S rRNA sequence was analyzed using BLAST in the NCBI database, and a phylogenetic tree was constructed using MEGA 7.0. The results showed that the 16S rRNA gene sequence of J8M8 had high similarity to B. subtilis (Figure S5). Subsequently, physiological and biochemical analyses were conducted on the J8M8 strain. The metabolic characteristics of this strain are consistent with the taxonomic descriptions of B. subtilis in Bergey’s Manual of Determinative Bacteriology. Specifically, the strain was positive for the Voges-Proskauer (V-P) test, nitrate reduction, starch hydrolysis, gelatin liquefaction, citrate utilization, and the fermentation of D-xylose, L-arabinose, and D-mannitol. It also showed robust growth in 7% NaCl and at pH 5.7. Crucially, the strain was negative for anaerobic growth and propionate utilization, effectively distinguishing it from closely related species such as Bacillus licheniformis (Table S3). These results confirm the identity of the J8M8 strain as B. subtilis. B. subtilis is recognized as a safe microorganism by the U.S. Food and Drug Administration. This FDCA-producing strain was designated B. subtilis J8M8, and its 16S rDNA sequence is listed in Table S4.
The safety of B. subtilis J8M8 was assessed by examining its biochemical pathogenicity markers. The results showed that B. subtilis J8M8 exhibited a γ-hemolysis on blood agar, in contrast to the prominent β-hemolysis produced by B. cereus (Figure S6a). Moreover, B. subtilis J8M8 exhibited no lecithinase activity, as evidenced by the absence of a precipitation halo on egg yolk agar, in contrast to B. cereus, which displayed a dense opalescent zone resulting from lecithin hydrolysis (Figure S6b). In conclusion, the absence of hemolytic and lecithinase activities in B. subtilis J8M8 indicates that it lacks key extracellular virulence factors common in pathogenic Bacillus species, thereby confirming its biological safety at the phenotypic level [29].

3.2. Optimization of Whole-Cell Catalytic Conditions for FDCA Production by B. subtilis J8M8

Generally, a higher cell concentration results in a greater amount of intracellular enzymes available for whole-cell catalysis, thereby enhancing FDCA production. However, excessively high cell densities can increase the viscosity of the reaction system, negatively affecting overall reaction performance [30]. Therefore, cell concentration was optimized to balance enzymatic activity and production. The results showed that FDCA production increased gradually with rising cell concentration, reaching a maximum of 28.4 mM at an OD600 = 90 (Figure 1a). Beyond this point, further increases in cell concentration did not significantly enhance FDCA production, possibly due to reduced oxygen transfer caused by higher viscosity [31]. Therefore, an OD600 of 90 was selected as the optimal cell concentration for FDCA production.
Whole-cell catalysis was also performed at varying HMF concentrations to evaluate its effect on FDCA production. FDCA production gradually increased as the HMF concentration was raised from 20 to 80 mM (Figure 1b). The highest FDCA yield (29.9 mM) was obtained at 80 mM HMF. However, further increases in HMF concentrations led to a significant decrease in FDCA production, likely due to the substrate inhibition or cytotoxic effects of high HMF concentrations [32]. Therefore, the optimal concentration of HMF was determined to be 80 mM.
FDCA production was measured at different pH values to determine the optimal pH for FDCA production via whole-cell catalysis. The maximum FDCA produced was 31.7 mM in phosphate buffer at pH 7.5 (Figure 1c). In whole-cell catalysis, the stability and activity of intracellular enzymes are strongly influenced by environmental pH, and they exhibit optimal catalytic activity only within a specific pH range [33]. Therefore, a pH of 7.5 was selected for whole-cell catalysis.
During biocatalysis, intracellular enzyme activity is highly sensitive to temperature, which can impact catalytic efficiency [34]. Examining the effects of temperature on FDCA production using whole-cell catalysis revealed that the maximum FDCA was obtained (33.1 mM) when the temperature was 35 °C (Figure 1d). This outcome is likely due to enzyme inactivation at excessively high temperatures, which reduces catalytic efficiency. Therefore, 35 °C was chosen as the optimal temperature for whole-cell catalysis.

3.3. Overexpression of HmfH, ADH, and MaAAO to Improve FDCA Production

Although we successfully achieved whole-cell synthesis of FDCA from HMF under optimal conditions using B. subtilis J8M8, the FDCA production remained relatively low. This was attributed to the accumulation of the intermediate product, HMFCA (38.4 mM, Figure 2a). Therefore, the conversion of HMFCA into FDCA represents the rate-limiting step in efficient FDCA synthesis [35]. Enhancing the expression of enzymes that can oxidize HMFCA is an effective strategy for improving the overall efficiency of FDCA synthesis. HmfH can continuously catalyze the oxidation of HMF to FDCA via HMFCA. For example, when HmfH was introduced into P. putida S12, a high FDCA production with a yield of 97% was achieved [36]. ADH has been reported to oxidize both HMFCA and 2,5-diformylfuran (DFF) [37]. Furthermore, MaAAO can oxidize HMFCA to FFCA [38]. Therefore, HmfH, ADH, and MaAAO were overexpressed in B. subtilis J8M8 to assess their effects on FDCA production, respectively. The electrophoretic pattern reveals distinct protein bands aligning with the theoretical molecular weights of HmfH (62.2 kDa), ADH (64.8 kDa), and MaAAO (68.1 kDa), indicating successful expression of these proteins in B. subtilis J8M8 (Figure S7). As shown in Figure 2b–d, FDCA production by strain BS-H (overexpressing HmfH), BS-A (overexpressing ADH), and BS-M (overexpressing MaAAO) reached 49.5 mM, 42.5 mM, and 39.3 mM at 84 h, respectively. Meanwhile, the accumulation of the intermediate product HMFCA was significantly reduced compared to the original strain (Figure S8).
To further improve the efficiency of FDCA synthesis, whole-cell catalysis using recombinant strains BS-HA (co-expressing HmfH and ADH), BS-HM (co-expressing HmfH and MaAAO), and BS-AM (co-expressing ADH and MaAAO) was performed to assess their FDCA production capacity (Figure S7). After 84 h of whole-cell catalysis, FDCA production reached 57.2 mM, 55.3 mM, and 51.5 mM for strains BS-HA, BS-HM, and BS-AM, respectively (Figure 3a–c). However, the intermediate product, HMFCA, still accumulated. To address this, the genes encoding HmfH, ADH, and MaAAO were co-expressed in a single strain to obtain BS-HAM (Figure S7). FDCA production by BS-HAM was increased to 72.3 mM, with a yield of 90.4% (Figure 3d). Notably, HMFCA accumulation was significantly reduced with BS-HAM (only 3.5 mM at 84 h), suggesting that co-expression of all three enzymes promoted the catalytic oxidation of HMFCA and improved the overall efficiency of FDCA synthesis.

3.4. Verification of the Oxidation Rate of HMFCA for FDCA Production

To further investigate the oxidation capacity of different recombinant strains toward HMFCA, a whole-cell catalysis experiment using HMFCA as the substrate was conducted. The results showed that the oxidation rates of HMFCA in all seven recombinant strains were increased compared with the wild-type strain (Figure 4). The BS-HAM strain exhibited the highest oxidation rate for HMFCA, reaching 1.52 mmol·L−1·h−1, which was 3.23 times higher than that of the wild-type strain. These findings indicate that overexpression of the target genes significantly enhances the strain’s ability to oxidize the intermediate HMFCA, thereby promoting its conversion to FDCA and providing direct experimental support for optimizing whole-cell catalysis of FDCA production from HMF.

3.5. Optimization of Whole-Cell Catalytic Conditions for FDCA Production by the BS-HAM Strain

Catalytic conditions for the recombinant strain can influence protein folding, stability, and activity. Therefore, re-optimization of these conditions was necessary to ensure that the enzymes exhibit maximal catalytic efficiency under the new physiological conditions [39]. First, cell concentration was optimized, and the highest FDCA production of 72.5 mM was achieved at an OD600 of 90 (Figure 5a). Next, the HMF concentration was optimized, with a maximum FDCA production of 78.7 mM obtained at 90 mM HMF (Figure 5b). The pH was then optimized, and the highest FDCA production of 83.1 mM was achieved at pH 7.0 in phosphate buffer (Figure 5c). Finally, temperature optimization revealed that the maximum FDCA production of 83.3 mM was at 35 °C (Figure 5d). Overall, through the optimization of whole-cell catalytic conditions, the key parameters for FDCA production by the BS-HAM strain were established. The final FDCA production reached 83.3 mM with a yield of 92.6%, and the intermediate product HMFCA was significantly reduced, demonstrating efficient FDCA biosynthesis.

4. Conclusions

Biological processes for producing FDCA are gaining increasing attention as environmentally friendly and sustainable alternatives. Whole-cell catalysis shows promise for the production of sustainable, bio-based platform compounds. In this study, we isolated a safe strain, B. subtilis J8M8, which catalyzed the production of FDCA from HMF. By enhancing the oxidation of the intermediate HMFCA and optimizing the whole-cell catalytic conditions, the final FDCA production reached 83.3 mM, with a yield of 92.6%. This study explored the biosynthesis of FDCA using safe bacteria, demonstrating their potential for future biotechnological applications.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms14020425/s1, Table S1: Primers used in this study; Table S2: The amino acid and nucleotide sequences of HmfH, ADH, and MaAAO; Table S3: Physiological and biochemical identification results of B. subtilis J8M8; Table S4: 16s rDNA sequence of B. subtilis J8M8; Figure S1: Map of the expression plasmids. (a) Map of the original plasmid pSTOP. (b) Map of the tandem genes expression plasmid pSTOP-hmfH-adh-MaAAO; Figure S2: (A) HPLC analysis on the standard of FDCA and its derivatives. (B) HPLC analysis on the fermentation supernatant of the J8M8 strain. (C) Compare the production of FDCA among three specifically selected strains; Figure S3: Schematic illustrating the process of synthesizing FDCA from HMF. HMF, 5-hydroxymethylfurfural; HMFCA, 5-hydroxymethyl-2-furancarboxylic acid; DFF, 2,5-diformylfuran; FFCA, 5-formyl-2-furancarboxylic acid; FDCA, 2,5-furandicarboxylic acid; Figure S4: The colony of B. subtilis J8M8 on agar plate; Figure S5: Phylogenetic tree based on the 16s rRNA gene sequence of B. subtilis J8M8; Figure S6: (a) Examination of hemolytic activity on blood agar plates in Bacillus cereus (left) and B. subtilis J8M8 (right). (b) Examination of lecithinase activity on egg yolk agar plates in Bacillus cereus (left) and B. subtilis J8M8 (right); Figure S7: SDS-PAGE analysis of the expression of various recombinant enzymes in B. subtilis J8M8. Lane M: protein marker; NC: negative control; Lane 1: BS-H strain; Lane 2: BS-A strain; Lane 3: BS-AM strain; Lane 4: BS-HM strain; Lane 5: BS-HA strain; Lane 6:BS-HAM strain. Lane 7: BS-M strain; Figure S8: HPLC spectra of FDCA and its derivatives from different B. subtilis strains. (a) Wild-type B. subtilis J8M8 under optimized conditions; (b–h) Whole-cell biocatalysis for FDCA production by BS-H, BS-A, BS-M, BS-HA, BS-HM, BS-AM, and BS-HAM strains, respectively.

Author Contributions

X.J.: Writing—original draft, Data curation. C.C.: Validation. M.J.: Data curation. J.H.: Methodology. X.H.: Investigation. T.W.: Writing—review and editing, Funding acquisition. D.H.: Software. Y.J.: Writing—review and editing. H.L.: Methodology. H.Y.: Conceptualization, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (22308180); the Key R&D Program of Shandong Province, China (2024CXGC010914); and Technology Innovation Guidance Program of Shandong Province (Central Guidance Fund for Local Science and Technology Development) (YDZX2024052).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and the Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

Author Xuechun Han is affiliated with Luwei Pharmaceutical Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Microorganisms 14 00425 g001
Figure 1. Optimization of whole-cell catalytic conditions for FDCA production by B. subtilis J8M8. (a) Effect of cell concentration on FDCA synthesis; (b) Effect of HMF concentration on FDCA synthesis; (c) Effect of pH on FDCA synthesis; (d) Effect of temperature on FDCA synthesis.
Figure 1. Optimization of whole-cell catalytic conditions for FDCA production by B. subtilis J8M8. (a) Effect of cell concentration on FDCA synthesis; (b) Effect of HMF concentration on FDCA synthesis; (c) Effect of pH on FDCA synthesis; (d) Effect of temperature on FDCA synthesis.
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Figure 2. (a) Whole-cell catalytic process for FDCA production by B. subtilis J8M8 under optimized conditions; (b) Effect of HmfH expression on FDCA production; (c) Effect of ADH expression on FDCA production; (d) Effect of MaAAO expression on FDCA production.
Figure 2. (a) Whole-cell catalytic process for FDCA production by B. subtilis J8M8 under optimized conditions; (b) Effect of HmfH expression on FDCA production; (c) Effect of ADH expression on FDCA production; (d) Effect of MaAAO expression on FDCA production.
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Figure 3. (a) Effect of co-expression of HmfH and ADH on FDCA production; (b) Effect of co-expression of HmfH and MaAAO on FDCA production; (c) Effect of co-expression of ADH and MaAAO on FDCA production; (d) Effect of co-expression of HmfH, ADH, and MaAAO on FDCA production.
Figure 3. (a) Effect of co-expression of HmfH and ADH on FDCA production; (b) Effect of co-expression of HmfH and MaAAO on FDCA production; (c) Effect of co-expression of ADH and MaAAO on FDCA production; (d) Effect of co-expression of HmfH, ADH, and MaAAO on FDCA production.
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Figure 4. Oxidation rates of HMFCA by different recombinant strains using HMFCA as the substrate.
Figure 4. Oxidation rates of HMFCA by different recombinant strains using HMFCA as the substrate.
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Figure 5. Optimization of whole-cell catalytic conditions for FDCA production by the recombinant strain BS-HAM. (a) Effect of cell concentration on FDCA synthesis; (b) Effect of HMF concentration on FDCA synthesis; (c) Effect of pH on FDCA synthesis; (d) Effect of temperature on FDCA synthesis.
Figure 5. Optimization of whole-cell catalytic conditions for FDCA production by the recombinant strain BS-HAM. (a) Effect of cell concentration on FDCA synthesis; (b) Effect of HMF concentration on FDCA synthesis; (c) Effect of pH on FDCA synthesis; (d) Effect of temperature on FDCA synthesis.
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Table 1. Plasmids and strains used in this study.
Table 1. Plasmids and strains used in this study.
Plasmids and StrainsCharacteristicSource
Plasmids
pSTOPAmp, Kan, PxylA promoter, used for gene expressionLab stock
pUC57Amp, used for gene cloneLab stock
pSTOP-hmfHPxylA promoter, expressing HmfHThis study
pSTOP-adhPxylA promoter, expressing ADHThis study
pSTOP-MaAAOPxylA promoter, expressing MaAAOThis study
pSTOP-hmfH-adhPxylA promoter, expressing HmfH and ADHThis study
pSTOP-hmfH-MaAAOPxylA promoter, expressing HmfH and MaAAOThis study
pSTOP-adh-MaAAOPxylA promoter, expressing ADH and MaAAOThis study
pSTOP-hmfH-adh-MaAAOPxylA promoter, expressing HmfH, ADH, and MaAAOThis study
Strains
B. subtilis J8M8Wild-type strainThis study
BS-HB. subtilis J8M8, containing pSTOP-hmfHThis study
BS-AB. subtilis J8M8, containing pSTOP-adhThis study
BS-MB. subtilis J8M8, containing pSTOP-MaAAOThis study
BS-HAB. subtilis J8M8, containing pSTOP-hmfH-adhThis study
BS-HMB. subtilis J8M8, containing pSTOP-hmfH-MaAAOThis study
BS-AMB. subtilis J8M8, containing pSTOP-adh-MaAAOThis study
BS-HAMB. subtilis J8M8, containing pSTOP-hmfH-adh-MaAAOThis study
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MDPI and ACS Style

Jiang, X.; Chen, C.; Jiao, M.; He, J.; Han, X.; Wang, T.; Huang, D.; Jiang, Y.; Liu, H.; Yuan, H. Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural by Bacillus subtilis J8M8. Microorganisms 2026, 14, 425. https://doi.org/10.3390/microorganisms14020425

AMA Style

Jiang X, Chen C, Jiao M, He J, Han X, Wang T, Huang D, Jiang Y, Liu H, Yuan H. Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural by Bacillus subtilis J8M8. Microorganisms. 2026; 14(2):425. https://doi.org/10.3390/microorganisms14020425

Chicago/Turabian Style

Jiang, Xiangling, Changtong Chen, Mingxia Jiao, Jianqi He, Xuechun Han, Tengfei Wang, Di Huang, Yi Jiang, Hongling Liu, and Haibo Yuan. 2026. "Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural by Bacillus subtilis J8M8" Microorganisms 14, no. 2: 425. https://doi.org/10.3390/microorganisms14020425

APA Style

Jiang, X., Chen, C., Jiao, M., He, J., Han, X., Wang, T., Huang, D., Jiang, Y., Liu, H., & Yuan, H. (2026). Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural by Bacillus subtilis J8M8. Microorganisms, 14(2), 425. https://doi.org/10.3390/microorganisms14020425

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