Aspergillus niger VT-2 (CICC2422), obtained from Hongying Biotechnology Co., Ltd. (Hunan, China), was cultured on potato dextrose agar (PDA) at 30°C. Escherichia coli TOP10 was used for all the molecular cloning experiments. DNA polymerase (PrimeSTAR™ HS), restriction enzymes, and T4 DNA ligase were purchased from Takara Bio, Inc. (Dalian, China). Oligonucleotides were synthesized by Sangon Biotech (Shanghai, China), and multiple DNA fragments were assembled via NEBuilder^® HiFi DNA Assembly Master Mix (Catalog No. E2621S). Reagents were obtained from Sigma-Aldrich (Sigma-Aldrich Co.). Unless otherwise specified, all chemicals were of analytical grade. Details of the strains, plasmids, and primers used in this study are available in Supplementary Tables S1–S3.

Ten catalases were identified from thermophilic or mesophilic fungi, including AnCat from Aspergillus niger (NCBI: AAA68206.1), TtCat from Thermothelomyces thermophilus (NCBI: XP_003663032.1), MtCat from Mycothermus thermophilus (NCBI: PP951965), TaCat from Thermoascus aurantiacus (NCBI: PP951966), PvCat from Paecilomyces variotii (NCBI: GAD95766.1), AfCat from Aspergillus fumigatus (NCBI: XP_748550.1), PmCat from Penicillium marneffei (NCBI: EEA19216.1), NcCat from Neurospora crassa (NCBI: XP_957826.1), ReCat from Rasamsonia emersonii (NCBI: XP_013328798.1), and TrCat from Talaromyces rugulosus (NCBI: XP_035343757.1). The nucleotide sequences of AnCat, TtCat, MtCat, TaCat, PvCat, AfCat, PmCat, NcCat, ReCat, and TrCat were obtained through gene synthesis by Shanghai Sangon Biotech, with synthetic gene fragments flanking 30 bp homology arms on both sides to the expression vector pANEXP. Using the NEBuilder^® HiFi DNA Assembly method, we then seamlessly connected the XbaI-linearized pANEXP vector with the synthesized fragments, forming the corresponding ten pANEXP-CAT expression vectors (pANEXP-ANCAT, pANEXP-TTCAT, pANEXP-MTCAT, pANEXP-TACAT, pANEXP-PVCAT, pANEXP-AFCAT, pANEXP-PMCAT, pANEXP-NCCAT, pANEXP-RECAT, and pANEXP-TRCAT).

Following the protocol established by Wernars et al. (1986), we prepared and transformed the protoplasts of the A. niger strain VT-002. We linearized the ten plasmids via Bgl II (Thermo Scientific, Waltham, MA, USA) and then transformed them into A. niger VT-2 through protoplast-mediated transformation. After transformation, we spread the transformed protoplasts on potato dextrose agar (PDA) plates supplemented with 1 M sorbitol and hygromycin B (300 μg/mL).

From the transformants of each catalase, 200 colonies were selected and transferred to 24-well deep-well plates, with each well containing 2 mL potato dextrose broth (PDB), and incubated for four days. After initial screening, the top six strains with the highest enzyme activity from each catalase transformant were chosen for further screening in a 24-well plate. The strain with the highest activity from each catalase group was subsequently selected for shake flask fermentation, which was conducted in three flasks. The strains with the highest enzyme activity were identified using the primers for the Pgla-fw/Tter-rw PCR. The harvested supernatants were centrifuged and filtered to measure catalase activity, and protein expression in the fermentation broth was analyzed via SDS–PAGE. Stability analyses under application conditions (45°C, pH 5.5) were conducted for each sample, with enzyme activity tested every 8 hours to assess stability under these conditions.

In this study, the pANEXP-TACAT vector was digested with SacI and XbaI, and the dual enzyme-cutting vector was recombined with synthesized long primers (S2, S3, S4, and S5 signal peptide-fw in Supplementary Table S3) via homologous recombination to replace the original S1 signal peptide. The S2, S3, S4, and S5 signal peptides were then inserted into the pANEXP-TACAT vector, transformed into A. niger for selection, and subjected to enzyme activity assays and SDS–PAGE analysis. Details of the high-enzyme activity screening process are described in Section 2.2.

The assembly of the heme-critical enzyme overexpression vector proceeded as follows: expression cassettes for Pamy+HemeA+TrpC and Ptef1+HemeB+TrpC were constructed via fusion PCR. These were then seamlessly linked with the amdS (GenBank: M16371.1) segment and the PUC57 vector fragment via the NEBuilder^® HiFi DNA Assembly method and transformed into Escherichia coli TOP10 competent cells, yielding the final construct, the pAN-amdS-hemeA+hemeB vector. The correct vector was identified via PCR. Following NotI digestion and transformation with the strain with the highest enzyme activity, AN-S5-TaCat, high-enzyme-activity strains were selected and analyzed for enzyme activity and heme content. The details of this process are described in Section 2.2.

The inoculum was spread onto 9 cm petri dishes containing 20 mL of PDA agar medium and incubated at 30°C for 4–5 days. Subsequently, 6–8 A. niger colonies were selected and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of CD medium (sucrose 30 g/L, NaNO₃ 2 g/L, K₂HPO₄ 1 g/L, MgSO₄ 0.5 g/L, KCl 0.5 g/L, FeSO₄ 0.01 g/L, pH 7.3), which were then cultured statically at 30°C for 72 hours. Afterward, 30 mL of the mycelial culture grown in CD medium was transferred into five separate 500 mL Erlenmeyer flasks, each containing 200 mL of liquefied starch medium. The cultures were incubated at 30°C with shaking at 200 rpm for 3 days. The liquefied starch medium consisted of 45 g/L liquefied corn starch, 0.04 g/L calcium chloride, 28 g/L corn steep liquor, and 10.0 g/L soybean meal. Pre-liquefaction of the corn starch was achieved using high-temperature amylase.

Following the shake flask cultivation, 1000 mL of primary seed culture was inoculated into a 20-liter fermenter containing 10 L of liquefied starch medium. During fermentation, the temperature was maintained at 30–32°C, and the pH was controlled between 5.0 and 5.5 with the addition of 25% ammonia solution. Stirring speed was set to 500 rpm, and the aeration rate was 20 L/min. The residual sugar concentration in the fermentation broth was kept between 15 and 30 g/L. After 64 hours of culture, 4 L of seed culture from the 20 L fermenter was transferred into a 50 L fermenter containing 25 L of liquefied starch medium. Fermentation conditions were similar, with temperature controlled at 30–32°C and pH maintained at 5.0–5.5 using ammonia solution. The sugar concentration in the feed solution was approximately 450 g/L, and the residual sugar concentration in the fermentation broth was kept between 5 and 20 g/L by manual control. Stirring speed remained at 500 rpm, and the aeration rate was increased to 40 L/min. The sugar feeding began after 24 hours of fermentation when the residual sugar concentration dropped below 10 g/L. A constant feeding rate of 2–3 g/L/h was maintained to keep the residual sugar level between 5 and 25 g/L. Between 85 and 110 hours, the feeding rate was lowered to 1–2 g/L/h due to a decline in sugar consumption. After 110 hours, the feeding rate was increased to 2–3.5 g/L/h as sugar consumption accelerated. At 140 h, the feeding rate was again reduced to 1–2 g/L/h. The fermentation process was completed after 210 to 220 hours. Throughout the cultivation, catalase activity, wet weight, and residual sugar concentration were continuously monitored.

Catalase activity was measured following the method described by Thorup (Thorup et al., 1961). To determine the optimal temperature and pH, we performed enzyme assays across temperatures ranging from 25°C to 90°C and pH values ranging from 2.5 to 8.0. During pH stability tests, we mixed diluted enzyme solutions with citrate-phosphate buffers at various pH values. The pH stability was determined post-incubation at 25°C for 24 hours. For the pH stability tests, we mixed the diluted enzyme solutions with citrate–phosphate buffers at various pH values. Additionally, we evaluated the tolerance of recombinant catalase to different metal ions by testing concentrations of 5 mM for Ca²⁺, Mg²⁺, Na⁺, K⁺, Ni²⁺, Zn²⁺, Al³⁺, Co²⁺, Fe³⁺, Ag⁺, Fe²⁺, and Cu²⁺.

Glycosylation analysis was validated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein concentration in the supernatant was diluted to between 0.1 g/L and 0.5 g/L, combined with glycoprotein denaturing buffer (NEB, B1704S), boiled at 100°C for 10 min, and then incubated with Endo H (NEB, P0702S) at 37°C for 1 hour, followed by SDS–PAGE alongside untreated samples.

NetNGlyc (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/) was used to predict glycosylation sites. Thermostability analysis was conducted by measuring the heat tolerance of Endo H-treated and untreated rTaCat at 75°C, 80°C, and 90°C for 5 min. Additionally, the thermostabilities of rTaCat and rAnCat were compared after incubation for 1 h at 60°C, 65°C, and 70°C.

The protein structure modeling and molecular dynamics simulation analyses are as follows: initially, TaCat and AnCat were modeled, and side-chain structures were optimized via Alphafold2 (Jumper et al., 2021) and Rosetta relax (Nivón et al., 2013), followed by molecular dynamics simulations via the optimized structure models. All molecular dynamics simulations in this study were performed via the AMBER99SB-ILDN force field in Gromacs 2022.5 (Singh et al., 2018). The detailed simulation process is provided in Supplementary material S1.

The production of GA involves catalytic generation from glucose in a 20-liter reactor. A total of 10 L of 36% glucose solution was added to a 20 L bioreactor, along with approximately 1 mL of antifoaming agent, catalase, and glucose oxidase. For sodium gluconate production, catalase and glucose oxidase were added to the reaction mixture at final concentrations of 300000 U/mL (added amount 20 g/kg) and 15,000 U/mL (added amount 5 g/kg), respectively. The reaction proceeded at 45°C with a pH of 5.5 at 500 rpm under a pressure of 0.1 MPa. During this process, the dissolved oxygen and alkali addition were monitored, and samples were taken to test the residual sugar content.

In search of catalases with thermal stability, we identified 10 catalases from thermophilic or mesophilic fungi. Through sequence homology analysis, we constructed a phylogenetic tree for these 10 catalases via MEGA software (Figure 1A). We subsequently cloned these catalase genes into the A. niger expression vector pANEXP and transformed them into A. niger host protoplasts for integration into the A. niger genome (Figure 1B). SDS–PAGE analysis revealed that 7 of the catalases were successfully expressed in A. niger (Figure 1C), including catalase from Thermoascus aurantiacus (TaCat), catalase from Paecilomyces variotii (PvCat), catalase from Aspergillus fumigatus (AfCat), catalase from Penicillium marneffei (PmCat), catalase from Neurospora crassa (NcCat), catalase from Rasamsonia emersonii (ReCat), and catalase from Aspergillus niger (AnCat). Among them, the TaCat and PmCat transformants presented the highest enzyme activities in shake-flask fermentation, with 9240 U/mL and 12433 U/mL, respectively (Figure 1D). The enzyme activities of the other catalases ranged from 5000 to 8000 U/mL, while TtCat from Thermothelomyces thermophilus, MtCat from Mycothermus thermophilus, and TrCat from Talaromyces rugulosus showed little to no enzyme activity. Under simulated conditions of a sodium gluconate reaction (45°C, pH 5.0), TaCat exhibited superior thermal stability, maintaining 90.6% activity, whereas the stability of the other transformants remained below 72% (Figure 1E). TaCat was selected as the most promising candidate catalase due to the data showing its excellent enzyme activity and thermal stability.

To further increase the enzymatic activity of the catalase TaCat in A. niger, we increased production through signal peptide optimization and enhancement of the production of the catalase cofactor heme. The secretion levels of the recombinant protein are markedly influenced by the type of signal peptide used. For our research, we chose four frequently used signal peptides that exhibit high expression: S2, S3, S4, and S5. Specifically, S2 is the glucoamylase signal peptide of A. niger, S3 is the glucoamylase of Rasamsonia emersonii, S4 is the lipase signal peptide from A. niger, and S5 is the amylase signal peptide of A. niger. In this study, we replaced the original S1 signal peptide with a homologous recombination sequence, constructing S2, S3, S4, and S5 signal peptides into the pANEXP-TACAT vector for expression in A. niger. As illustrated in Figure 2A, the enzyme activities of recombinant strains modified with the S2, S4, and S5 signal peptides were significantly higher than those of the strains modified with the wild-type S1 signal peptide. Notably, the activities of the S2 and S5 peptides were 1.15 times and 1.29 times greater than those of the wild-type strains, respectively, and they substantially exceeded those of the control. Figure 2B demonstrates that the protein level of catalase expressed with the S5 signal peptide was higher than that expressed with the wild-type S1 signal peptide. These findings suggest that signal peptides derived from A. niger are effective for the expression of exogenous proteins in A. niger. Based on these results, AN-S5-TaCat was selected as the candidate for further investigation.

Heme is an essential cofactor in heme-dependent catalase (Pedersen et al., 2008; Maresca et al., 2018), and the conversion of apoprotein to heme protein depends on the use of heme provided by the heme biosynthesis pathway. As shown in Figure 3A, eight specific enzymes are involved in the biosynthesis of fungal heme; we focused on two key enzymes, HemeA and HemeB. Initially, by employing NEBuilder^® HiFi DNA Assembly, we assembled the expression cassettes for HemeA, HemeB, amdS, and fragments of the PUC57 vector into the complete vector pAN-amdS-HemeA+HemeB (shown in Figure 3B). Following linearization, the vector was transformed into the S5-TaCat protoplast strain of A. niger via protoplast transformation and plated onto CD+ acetamide plates. When the transformants were streaked onto PDA plates, the recombinant strain was reddish, whereas the background strain was grayish white (Figure 3C). As shown in Figure 3D, the enzymatic activity of the transformant AN-S5-TaCat-HemeA+HemeB substantially increased to 17761.3 U/mL, a 50.5% increase over that of AN-S5-TaCat. We also assessed the intracellular heme levels and detected an 88.1% increase in AN-S5-TaCat-HemeA+HemeB compared with AN-S5-TaCat.

To explore the industrial applicability of the newly constructed catalase strain AN-S5-TaCat-HemeAB, the production capacity of the engineered strain was determined by culturing it in a 50-L fermenter. As depicted in Figure 4, the enzymatic activity of the strain's catalase continuously increased during the entire 213-h fermentation process, finally reaching a high level of 321,779.5 U/mL. The increase in enzymatic activity followed an approximate S-shaped curve, with slow growth occurring between 0–93 hours and 164–213 hours and logarithmic growth occurring during the 93–164-hour period. In the fermenter, the engineered cells grew rapidly, reaching a peak wet cell weight of 610 mg-CWW/mL at 68 hours. Afterward, the wet weight began to decrease and fluctuated, ending at 490 mg-CWW/mL. Additionally, we observed that the mycelia gradually separated into smaller filaments at ~80 h (in Supplementary Figure S1). The high level of catalase activity and rapid growth indicate that the improved strain has substantial industrial application potential.

The enzymatic characteristics of catalase produced by the recombinant strain AN-S5-TaCat-hemeAB were studied. The catalase activity was measured within the range of 30–90°C, and as shown in Figure 5A, the optimal reaction temperature for rTaCat was 40°C, with the relative activity maintained above 75% between 25 and 75°C. The optimal pH for rTaCat is 6.0, with the relative activity remaining above 70% within a pH range of 4.0–9.0 (Figure 5B). Research on pH stability (Figure 5C) revealed that rTaCat remains relatively stable from pH 4.0 to 10.0, preserving more than 80% of its enzymatic activity after 8-h incubation. This property of the catalase is advantageous for its use in the sodium gluconate conversion process, which generally occurs under acidic conditions (pH 5.0–6.0). Additionally, most of the residual chemicals and ions detected in this study had a negative impact on the enzymatic activity of rTaCat (Figure 5D). Na⁺, Ca²⁺, and K⁺ had little inhibitory effect on the activity of the enzyme. The enzyme retained only approximately 30% and 40% of its activity in the presence of Cu²⁺ and Co²⁺, respectively. It is speculated that the metal ions form coordination bonds with key amino acids of the rTaCat catalase, altering its conformation and thus affecting enzyme activity.

The theoretical molecular weight of rTaCat is presumed to be ~90 kDa. Nonetheless, SDS–PAGE (Figure 6A) demonstrated that the molecular weight of rTaCat expressed in A. niger exceeds 100 kDa, surpassing the theoretical estimate. Given the glycosylation system in A. niger, we speculate that this band may represent the glycosylated state. To verify this hypothesis, rTaCat was digested with the endoglycosidase Endo H, which removes nearly all N-linked oligosaccharides from the glycoprotein. We observed that the band for the rTaCat protein decreased in size after Endo H treatment, with a molecular weight of ~90 kDa (Figure 6A), which is consistent with the theoretical molecular weight. Glycosylation prediction revealed 7 potential glycosylation sites (Figure 6B). Among these potential glycosylation sites, N62, N131, and N562 exhibit high signal N-glycosylation. Furthermore, the thermal stability of both rTaCat and its deglycosylated form was assessed. As shown in Figure 6C, the deglycosylated catalase slightly decreased in thermal stability at 75°C and 85°C, but markedly decreased after heating at 90°C, with retention ranging from 38% to 15%.

We compared the high temperature tolerances of rTaCat and rAnCat produced from the engineered strains. The catalase rAnCat, derived from A. niger, is a commonly used commercial product. Figure 6D shows that, compared with rAnCat, rTaCat has notably greater heat resistance. Throughout the temperature range tested, rTaCat demonstrated superior thermal stability, maintaining near or exceeding 80% relative activity between 60°C and 70°C and retaining 49.7% activity even after one hour at 80°C. In contrast, rAnCat has virtually no activity above 70°C, highlighting its extreme instability at high temperatures.

We predicted the three-dimensional structures of TaCat and AnCat via AlphaFold2 and performed accelerated molecular dynamics simulations on the optimal model structures. Using AlphaFold2, we predicted the three-dimensional protein structures of the TaCat and AnCat catalases, obtaining highly accurate pLDDT scores of 93.13 and 93.02, respectively. Selecting the optimal model structures as initial conformations, we conducted 50 ns of accelerated molecular dynamics simulations on these two enzymes. We explored the theoretical differences in the stability and influencing factors of the two types of catalases, as shown in Figures 7A, B. Over three simulations, the root mean square deviation (RMSD) indicated protein system stability, with TaCat consistently showing a lower RMSD than AnCat with more stable fluctuations, suggesting superior structural stability for TaCat. Moreover, by measuring the radius of gyration (Rg), we found that the TaCat structure was more compact than the AnCat structure was, further implying its greater stability (Figures 7A, B). TaCat also displayed a more extensive hydrogen bond network than AnCat across the simulations, which was often linked to improved thermostability (Figures 7E, F). The root mean square fluctuation (RMSF) represents the average change in the atomic position over time, indicating the flexibility and intensity of protein amino acid movements throughout the simulation. Supplementary Figure S2 shows notable fluctuations in the TAcat protein between the regions of 1–100 aa and 400–490 aa. If further improvements in thermal stability are needed, focusing on studying and mutating this region will be our priority.

To demonstrate the practicality of rTaCat catalase in the biocatalytic production of gluconic acid (GA), a sodium gluconate application test was conducted in a 20 L bioreactor. The reaction conditions were maintained at 45°C and pH 5.5, with a stirring speed of 500 rpm and a pressure of 0.1 MPa. The results, detailed in Table 1, highlight the performance differences between TaCat and AnCat. Under identical conditions with the same quantity of glucose oxidase (GOD) added, TaCat demonstrated rapid kinetics, achieving complete conversion of glucose to GA in an average of 22 hours across three trials. In contrast, AnCat exhibited slower reaction kinetics, with an incomplete reaction observed after an average of 30 hours in three trials. Collectively, these findings underscore the superior efficacy of TaCat in sodium gluconate production compared with that of the conventional catalase AnCat.