Fusion myo-inositol oxygenase for simultaneously improving soluble expression and catalytic activity and application thereof

Abstract

The application discloses a fusion myo-inositol oxygenase for simultaneously improving soluble expression and catalytic activity and application thereof. The fusion myo-inositol oxygenase is composed of an NusA tag fused to the N terminal of myo-inositol oxygenase (Miox). Compared with wild-type Miox, the soluble expression level of the fusion myo-inositol oxygenase is significantly improved, and the specific enzyme activity reaches 0.98 U / mg, which is 48.5% higher than that of the wild type. The recombinant strain carrying the fusion myo-inositol oxygenase is used for biosynthesis of D-glucaric acid, and the yield of shake flask fermentation reaches 286.5 mg / L, which is 31.9% higher than that of the wild type. The fusion myo-inositol oxygenase provided by the application breaks through the limitation that traditional fusion tags are difficult to simultaneously consider expression and activity, and provides an effective technical scheme for efficient biosynthesis of D-glucaric acid.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, enzyme engineering, and metabolic engineering, specifically to an inositol oxygenase fusion protein that significantly enhances soluble expression and catalytic activity by fusing a soluble tag, its encoding gene, recombinant vector, recombinant strain, and its application in the biosynthesis of D-gluconic acid. Background Technology

[0002] D-Glucaric acid is a high-value-added dicarboxylic organic acid widely used in chemical, food, and pharmaceutical industries. In 2004, D-Glucaric acid was listed as one of the twelve "most valuable biorefined products." Microbial fermentation synthesis of D-Glucaric acid is gradually becoming an important alternative to chemical synthesis due to its mild reaction conditions, high selectivity, and green sustainability.

[0003] In the D-gluconic acid biosynthesis pathway constructed in *E. coli*, myo-inositoloxygenase (Miox) is the key rate-limiting enzyme. Its catalytic activity is one order of magnitude lower than that of inositol-1-phosphate synthase (Ino1) in the same pathway, and several orders of magnitude lower than that of uronic acid dehydrogenase (Udh). Studies have shown that the yield of D-gluconic acid is significantly linearly positively correlated with the activity of Miox. Therefore, improving the catalytic efficiency of Miox is the core strategy for enhancing the biosynthetic capacity of D-gluconic acid.

[0004] However, Miox exhibits low soluble expression levels and poor stability in E. coli expression systems. Wild-type Miox readily forms inclusion bodies during heterologous expression, resulting in low yields of catalytically active soluble proteins and severely limiting the overall efficiency of the D-gluconic acid synthesis pathway. Therefore, effectively improving the soluble expression level and catalytic activity of Miox is a key technical problem urgently needing to be solved in this field.

[0005] Fusion tagging technology is a common strategy for enhancing the soluble expression of recombinant proteins. Currently used fusion tags include glutathione S-transferase (GST), small ubiquitin-associated modified protein (SUMO), disulfide bond-forming protein A (DsbA), maltose-binding protein (MBP), N-utilization substance A (NusA), and green fluorescent protein (GFP). However, the effects of fusion tags on the structure and function of target proteins are unpredictable, and different tags have significantly different effects on the solubilization and activity of the same target protein. While some tags can effectively inhibit protein aggregation and improve soluble expression, they may cause conformational distortion of the active site due to steric hindrance, resulting in inactive soluble proteins. Therefore, for specific target proteins, systematic screening is often required to determine the optimal fusion tag, and the results of this screening cannot be reasonably predicted from existing technologies. Summary of the Invention

[0006] To overcome the technical bottleneck of traditional fusion tags' inability to simultaneously enhance the soluble expression and catalytic activity of inositol oxygenase (Miox), this invention systematically evaluated the effects of six commonly used soluble tags—NusA, SUMO, GST, DsbA, MBP, and GFP—on Miox protein expression and activity for the first time. Surprisingly, among the tested tags, only the NusA tag exhibited the best overall performance. Its fusion protein, NusA-Miox, not only showed a significantly higher soluble expression level than other tags, but also achieved an enzyme activity of 0.98 U / mg, a 48.5% increase compared to wild-type Miox. While the GST tag significantly enhanced enzyme activity, the soluble expression band could not be clearly distinguished from the co-migrating Ino1 protein. The SUMO tag showed a good enhancement in soluble expression, but its enzyme activity enhancement was less significant than that of the NusA tag. The DsbA, MBP, and GFP tags failed to simultaneously achieve a dual enhancement of both soluble expression and enzyme activity.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A first aspect of the present invention provides a fusion inositol oxygenase that simultaneously enhances soluble expression and catalytic activity, the fusion inositol oxygenase being formed by fusing a soluble tag with an inositol oxygenase Miox; the soluble tag being selected from one of NusA, GST, SUMO, DsbA, MBP, and GFP;

[0009] Furthermore, the soluble label is NusA, which is located at the N-terminus of inositol oxygenase Miox;

[0010] Furthermore, the amino acid sequence of the fused inositol oxygenase is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0011] A second aspect of the present invention provides a gene encoding the above-described fusion inositol oxygenase, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0012] A third aspect of the present invention provides a recombinant vector containing the aforementioned genes.

[0013] A fourth aspect of the present invention provides a recombinant strain containing the above-described recombinant vector;

[0014] Furthermore, the host of the recombinant strain is Escherichia coli, and it also co-expresses inositol-1-phosphate synthase Ino1, methanol dehydrogenase Mdh, 3-hexose-6-phosphate synthase Hps, 6-phosphate-3-hexose isomerase Phi, endogenous phosphatase Suhb, and uronic acid dehydrogenase Udh.

[0015] Furthermore, the *E. coli* strain in question is *E. coli* JM109(DE3) with the *frmRAB*, *rpiA*, *pfkA*, and *pfkB* genes knocked out.

[0016] The fifth aspect of the present invention provides a method for producing D-gluconic acid, wherein the above-mentioned recombinant strain is inoculated into a culture medium containing a carbon source for fermentation culture, and the expression of fused inositol oxygenase is induced to catalyze the production of D-gluconic acid from the substrate.

[0017] The sixth aspect of the present invention provides the application of the above-described fused inositol oxygenase in increasing the yield of D-gluconic acid.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) This invention systematically evaluated the effects of six commonly used fusion tags (NusA, SUMO, GST, DsbA, MBP, and GFP) on the soluble expression and catalytic activity of inositol oxygenase (Miox). The results showed that among the six tags tested, only the NusA tag could simultaneously achieve a significant increase in soluble expression level and a substantial increase in enzyme activity. Although the SUMO tag improved soluble expression to some extent, its enzyme activity increase was only 31.8%, significantly lower than the 48.5% increase of the NusA tag. The GST tag increased enzyme activity by 40.9%, but the soluble expression band could not be clearly distinguished from the co-expressed Ino1 protein (approximately 60 kDa); the DsbA tag enzyme activity showed no significant difference from the wild type; the MBP and GFP tags failed to improve enzyme activity, and the MBP tag actually reduced the soluble expression level of Miox. These screening results further highlight the unique superiority of NusA-Miox.

[0020] (2) SDS-PAGE analysis confirmed that NusA-Miox had significantly increased soluble expression levels in Escherichia coli compared with wild-type Miox and other fusion tags, with a distinct and specific protein band at the expected molecular weight (89 kDa).

[0021] (3) The enzyme activity assay results showed that the specific enzyme activity of NusA-Miox reached 0.98 U / mg, which was 48.5% higher than that of wild-type Miox (0.66 U / mg).

[0022] (4) The recombinant strain expressing NusA-Miox was used for the fermentation production of D-gluconic acid. The yield of shake-flask fermentation reached 286.5 mg / L, which was 31.9% higher than that of the control strain expressing wild-type Miox (217.1 mg / L), significantly improving the synthesis efficiency of the target product. Attached Figure Description

[0023] Figure 1 Electrophoresis images of the PCR amplification products of six fusion genes. A: PCR product of nusA-miox; B: PCR product of gst-miox; C: PCR product of sumo-miox; D: PCR product of dsbA-miox; E: PCR product of mbp-miox; F: PCR product of miox-gfp.

[0024] Figure 2 This is a schematic diagram of the structures of six fusion tag recombinant plasmids.

[0025] Figure 3 SDS-PAGE analysis results of different fusion protein inositol oxygenases show the soluble expression of wild-type Miox and six fusion tag modified inositol oxygenases in Escherichia coli. Lane M: Protein molecular weight standard (unit: kDa); Lane 1: Control strain EERC-2 (empty vector); Lane 2: Control strain EERC-5 (wild-type Miox, theoretical molecular weight approximately 33 kDa); Lane 3: Recombinant strain EERC-20 (NusA-Miox, theoretical molecular weight approximately 89 kDa); Lane 4: Recombinant strain EERC-21 (GST-Miox, theoretical molecular weight approximately 59 kDa); Lane 5: Recombinant strain EERC-22 (SUMO-Miox, theoretical molecular weight approximately 45 kDa); Lane 6: Recombinant strain EERC-23 (DsbA-Miox, theoretical molecular weight approximately 57 kDa); Lane 7: Recombinant strain EERC-24 (MBP-Miox, theoretical molecular weight approximately 80 kDa); Lane 8: Recombinant strain EERC-25 (Miox-GFP, theoretical molecular weight approximately 62 kDa).

[0026] Figure 4This figure compares the yields of D-gluconic acid synthesized by recombinant strains expressing different fusion inositol oxygenases under shake-flask fermentation conditions. EERC-16 is the control strain expressing wild-type Miox; EERC-26 to EERC-31 are recombinant strains expressing NusA-Miox, GST-Miox, SUMO-Miox, DsbA-Miox, MBP-Miox, and Miox-GFP, respectively. Detailed Implementation

[0027] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0028] The culture medium used in this invention has the following formulation:

[0029] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the remainder being water. LB solid medium is LB liquid medium with the addition of 1.5 wt% agar powder.

[0030] M9 medium: 12.8 g / L disodium hydrogen phosphate heptahydrate, 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 1 g / L ammonium chloride, 2 mM magnesium sulfate, 0.1 mM calcium chloride, and the remainder is water.

[0031] The pETDuet-ino1 vector involved in this invention is obtained by ligating the inositol-1-phosphate synthase gene ino1 (NCBI sequence number NP_012382.2) between the Nde I and Kpn I restriction sites of the pETDuet-1 vector.

[0032] The pRSFDuet-mdh-hps-phi plasmid involved in this invention is obtained by using the pETDuet-1 vector as the backbone vector, inserting the methanol dehydrogenase gene mdh (NCBI sequence number WP_013953014.1) between the Nco I and Hind III restriction sites, and sequentially inserting the 3-hexose-6-phosphate synthase hps (NCBI sequence number WP_014707664) and the 6-phosphate-3-hexose isomerase Phi (NCBI sequence number WP_064496767) between the Nde I and xho I restriction sites.

[0033] The pCDFDuet-subh-udh plasmid involved in this invention is obtained by inserting the endogenous phosphatase gene suhb (NCBI gene number 947285) between the NcoI and BamH I restriction sites and the uronic acid dehydrogenase gene udh (NCBI sequence number WP_011103399.1) between the Nde I and Kpn I restriction sites.

[0034] The EERC-15 involved in this invention is based on E. coli JM109(DE3) and uses λ-red homologous recombination to sequentially knock out the frmRAB gene (the frmRAB gene includes three genes: the transcriptional repressor gene frmR, the S-(hydroxymethyl)glutathione dehydrogenase gene frmA, and the S-formylglutathione hydrolase gene frmB; the NCBI gene number of frmR is 944986, the NCBI gene number of frmA is 944988, and the NCBI gene number of frmB is 944991, located on the E. coli genome f The rmR, frmA, and frmB are arranged in a continuous sequence. The λ-red homologous recombination method can knock out all frmR, frmA, and frmB at once without considering the influence of the knockout order. The ribose-5-phosphate isomerase A gene rpiA (NCBI gene number 947407), the 6-phosphofructokinase 1 gene pfkA (NCBI gene number 948412), and the 6-phosphofructokinase 2 gene pfkB (NCBI gene number 946230) are obtained.

[0035] Example 1: Construction of six fusion tag-Miox recombinant expression plasmids

[0036] Six commonly used soluble tags—NusA, GST, SUMO, DsbA, MBP, and GFP—were selected, and tag coding sequences optimized by *E. coli* codons were obtained through artificial gene synthesis. Overlap extension PCR was used to fuse each tag sequence with the inositol oxygenase encoding gene *miox*: the NusA, GST, SUMO, DsbA, and MBP tags were fused to the N-terminus of *miox*, and the GFP tag was fused to the C-terminus. Six fusion gene fragments were amplified, with expected lengths of: nusA-miox 2379 bp (SEQ ID NO. 1), gst-miox 1539 bp, sumo-miox 1182 bp, dsbA-miox 1536 bp, mbp-miox 2082 bp, and miox-gfp 1572 bp. After purification by 1.5% agarose gel electrophoresis, the band sizes were consistent with theoretical expectations. Figure 1 ).

[0037] Based on the restriction enzyme sites at both ends of the fusion fragment, the purified fusion fragment was double-digested with EcoRI / HindIII or SacI / SalI restriction endonucleases, respectively. Simultaneously, the pETDuet-ino1 vector was double-digested with the same restriction endonucleases to obtain a linearized vector. The digested fusion gene fragment and the linearized vector were mixed at a molar ratio of 3:1, and T4 DNA ligase was added, with ligation incubated overnight at 16°C. The six ligation products were transformed into E. coli Trans 10 competent cells using chemical transformation. The transformed bacterial culture was plated on LB agar containing 100 μg / mL ampicillin and incubated overnight at 37°C. The following day, 10–20 single colonies were randomly selected from each plate for colony PCR verification. Clones with positive PCR results were sequenced to confirm the complete correctness of the fusion gene sequence.

[0038] Six recombinant expression plasmids were successfully constructed: pETDuet-nusA-miox-ino1, pETDuet-gst-miox-ino1, pETDuet-sumo-miox-ino1, pETDuet-dsba-miox-ino1, pETDuet-mbp-miox-ino1, and pETDuet-miox-gfp-ino1. Figure 2 ).

[0039] Example 2: Induced expression of six fusion proteins and SDS-PAGE solubility analysis

[0040] The six recombinant expression plasmids constructed in Example 1 were transformed into E. coli JM109(DE3) competent cells by electroporation to obtain six recombinant strains, named EERC-20 to EERC-25, respectively. Two control strains were also constructed: Control strain EERC-2 was obtained by electroporating the empty vector pETDuet-1 into E. coli JM109(DE3) competent cells; Control strain EERC-5 was obtained by inserting the wild-type miox gene (NCBI sequence number: AAF25202.1) into the EcoRI and HindIII restriction sites of the pETDuet-ino1 vector to construct the recombinant plasmid pETDuet-miox-ino1, which was then electroporated into E. coli JM109(DE3) competent cells.

[0041] Each experimental bacterial strain was inoculated into LB liquid medium and activated overnight at 37°C and 250 rpm with shaking. The activated bacterial culture was then transferred to 200 mL of LB liquid medium at a 1 vol% inoculation rate and cultured at 37°C and 250 rpm for expansion. When the cell density in the shake flask reached OD0.05...600 When the concentration of β-carboxylic acid (β-C) reached 0.6, IPTG was added to a final concentration of 0.1 mM, and the culture conditions were adjusted to 30°C and 250 rpm, with induction continuing for 24 h. After induction, the culture medium was centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was discarded. The bacterial cell pellet was washed twice with pre-cooled 50 mM potassium phosphate buffer (pH 7.0) to thoroughly remove residual culture medium. The washed bacterial cells were resuspended in the same buffer. To ensure the comparability of experimental results, the OD of the cell suspension was measured using a microplate reader. 600 The OD value of the cell suspension for each sample is determined by adding or reducing the amount of buffer. 600 The value was uniformly adjusted to 2.7, and the final volume was set at 4 mL.

[0042] Take 4 mL of standardized cell suspension and sonicate it under ice bath conditions. The sonication parameters are: amplitude 30%, cycle time 2 s sonication / 2 s interval, total processing time 10 min. Centrifuge the disrupted sample at 4℃ and 8000 rpm for 5 min, and collect the supernatant as crude enzyme solution. Mix the crude enzyme solution with 4× protein loading buffer at a volume ratio of 3:1 and heat in a boiling water bath for 10 min to fully denature the protein. Load 15 μL of the denatured sample into a pre-prepared 12% separating gel and electrophoresis at 120V constant voltage for 60 min. After electrophoresis, rinse the gel with ultrapure water to remove residual electrophoresis buffer. Immerse the gel in Coomassie Brilliant Blue rapid staining solution, heat to boiling, and continue staining for 2 min. Then place the gel on a shaker and shake for 10 min, followed by multiple destaining with ultrapure water until the background is clear. Use a gel imaging system to record and analyze protein expression results.

[0043] Based on theoretical predictions, the molecular weights of each fusion protein are as follows: NusA-Miox 89kDa, GST-Miox 59kDa, SUMO-Miox 45kDa, DsbA-Miox 57kDa, MBP-Miox 80kDa, Miox-GFP 62kDa, and wild-type Miox 33kDa. SDS-PAGE results show (…). Figure 3Only NusA-Miox (lane 3, 89 kDa) and SUMO-Miox (lane 5, 45 kDa) showed distinct and specific bands at their expected molecular weight positions, indicating that these two fusion tags can significantly enhance the soluble expression of Miox. GST-Miox (lane 4, 59 kDa), DsbA-Miox (lane 6, 57 kDa), and Miox-GFP (lane 8, 62 kDa) co-migrated in the gel due to their molecular weight being close to that of the co-expressed Ino1 protein (approximately 60 kDa), resulting in indistinguishable bands. No distinct band was observed at the expected molecular weight position for MBP-Miox (lane 7, 80 kDa), suggesting that the MBP tag may induce conformational changes in the Miox protein, thereby reducing its soluble expression level.

[0044] Example 3: Determination of the specific enzyme activity of inositol oxygenase in six fusion proteins

[0045] Crude enzyme solutions were prepared from EERC-5 (wild-type control) and strains EERC-20 to EERC-25 using the same culture, induction, and cell treatment methods as described in Example 2.

[0046] The enzyme activity assay reaction system (total volume 1 mL) contained: 50 mM potassium phosphate buffer (pH 7.0), 2 mM L-cysteine, 1 mM ferrous ammonium sulfate, 60 mM inositol, and an appropriate amount of crude enzyme solution. Before the reaction, all components (including the crude enzyme solution) except for the substrate inositol were mixed and incubated at 30°C for 10 min to activate Miox. Inositol was then added to initiate the reaction, and the reaction was carried out accurately in a 30°C water bath for 1 h. After the reaction was completed, 1 / 10 volume of 30 wt% trichloroacetic acid was immediately added to terminate the reaction.

[0047] 40 mg of phenol and 5.4 mg of ferric chloride were dissolved in 10 mL of concentrated HCl to obtain a colorimetric reagent. An equal volume of the enzyme activity assay sample was mixed with twice the volume of the colorimetric reagent, and the mixture was heated in a boiling water bath for 30 min to initiate the colorimetric reaction. After the reaction was complete, the mixture was cooled to room temperature, and the absorbance was measured at 670 nm using a UV-Vis spectrophotometer. A reaction system without added inositol substrate was used as a blank control to eliminate background interference. The concentration of the product in the sample was calculated based on a pre-plotted D-glucuronic acid standard curve. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of D-glucuronic acid per minute. Specific enzyme activity is expressed as U / mg total protein.

[0048] Enzyme activity assays showed that the specific enzyme activity of wild-type Miox was 0.66 U / mg. The six fusion proteins exhibited differentiated enzyme activity: NusA-Miox had the highest specific enzyme activity, reaching 0.98 U / mg, a 48.5% increase compared to wild-type; GST-Miox and SUMO-Miox had specific enzyme activities of 0.87 U / mg and 0.93 U / mg, respectively, representing increases of 31.8% and 40.9% compared to wild-type; DsbA-Miox had a specific enzyme activity of 0.67 U / mg, with no significant difference from wild-type; MBP-Miox and Miox-GFP had specific enzyme activities of 0.64 U / mg and 0.65 U / mg, respectively, showing no significant increase.

[0049] Table 1. Results of specific enzyme activity assays for inositol oxygenase modified with different fusion tags.

[0050]

[0051] Example 4: Performance comparison of recombinant strains in synthesizing D-gluconic acid through shake-flask fermentation

[0052] The six recombinant expression vectors pETDuet-nusA-miox-ino1, pETDuet-gst-miox-ino1, pETDuet-sumo-miox-ino1, pETDuet-dsba-miox-ino1, pETDuet-mbp-miox-ino1, and pETDuet-miox-gfp-ino1 constructed in Example 1 were co-transformed with the plasmids pRSFDuet-mdh-hps-phi and pCDFDuet-subh-udh into the chassis strain EERC-15, respectively, to obtain recombinant strains EERC-26 to EERC-31 carrying the complete D-gluconic acid synthesis pathway. Meanwhile, the control strain EERC-16 was constructed: the recombinant plasmid pETDuet-miox-ino1 (carrying the wild-type miox gene, construction method is described in Example 2) and the plasmids pRSFDuet-mdh-hps-phi and pCDFDuet-subh-udh were transformed into the chassis strain EERC-15 using the same co-transformation method as the experimental group.

[0053] Each recombinant bacterial strain was inoculated into LB liquid medium and activated overnight at 37°C and 250 rpm with shaking. The activated bacterial solution was then inoculated into M9 medium with 9 g / L xylose as the sole carbon source at a 1 vol% inoculation rate and cultured at 37°C and 250 rpm. When the cells reached the logarithmic growth phase, they were collected by centrifugation and resuspended in fresh M9 medium, while simultaneously supplemented with 9 g / L xylose, 8 g / L methanol, and 0.1 mM IPTG. D-gluconic acid synthesis was induced at 30°C and 250 rpm, and fermentation was continued for 24 h.

[0054] After fermentation, the fermentation broth samples were pretreated: the samples were thoroughly mixed with Affi-Gel boric acid affinity gel and 0.1M potassium phosphate buffer (pH 7.0), and the gel was washed with a buffer solution composed of 80mM potassium phosphate and 20mM boric acid (pH 7.0). The target product was eluted with 0.1M HCl solution, and the eluent was collected and neutralized to pH 7.0 with 5M NaOH solution. All samples were centrifuged at 15000 rpm for 10 min to remove cell debris, and then filtered through a 0.22 μm filter membrane to remove impurities. Quantitative analysis was performed using a Hitachi high-performance liquid chromatography (HPLC) system under the following chromatographic conditions: Aminex HPX-87H column (300 × 7.8 mm), mobile phase: 5mM H₂SO₄ solution, flow rate: 0.6 mL / min, column temperature: 30℃, and UV detector: 210 nm. Each sample was analyzed in triplicate.

[0055] The results of shake-flask fermentation showed that ( Figure 4 The NusA-Miox fusion protein exhibited the best catalytic performance, enabling the EERC-26 strain to achieve a D-gluconic acid yield of 286.5 mg / L, a 31.9% increase compared to the control group EERC-16 (217.1 mg / L). The yield increases of GST-Miox (257.3 mg / L, an 18.5% increase) and SUMO-Miox (277.6 mg / L, a 27.8% increase) were both lower than those of NusA-Miox. The yield increase of DsbA-Miox (231.5 mg / L, a 6.6% increase) was limited. The yields of the MBP-Miox and Miox-GFP fusion protein strains did not show significant increases.

[0056] The fermentation results were highly consistent with the in vitro enzyme activity assay results of Example 3, further verifying the optimal performance of NusA-Miox fusion protein in the D-gluconic acid biosynthesis pathway.

[0057] Based on the above results, among the six commonly used fusion tags evaluated, the NusA tag is the only one that can simultaneously achieve a significant increase in Miox soluble expression levels and maximize enzyme activity. This invention demonstrates the unique superiority and non-obviousness of the NusA-Miox fusion protein, providing an effective technical solution for improving the biosynthetic efficiency of D-gluconic acid.

Claims

1. A fusion inositol oxygenase that simultaneously enhances soluble expression and catalytic activity, characterized in that: The fused inositol oxygenase is formed by fusing a soluble tag with inositol oxygenase Miox; the soluble tag is selected from one of NusA, GST, SUMO, and MBP.

2. The fused inositol oxygenase according to claim 1, characterized in that: The soluble tag is NusA, which is located at the N-terminus of inositol oxygenase Miox.

3. The fused inositol oxygenase according to claim 2, characterized in that: The amino acid sequence of the fused inositol oxygenase is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.

1.

4. A gene encoding the fusion inositol oxygenase according to any one of claims 2 to 3, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. A recombinant vector, characterized in that: The recombinant vector contains the gene described in claim 4.

6. A recombinant bacterial strain, characterized in that: The recombinant strain contains the recombinant vector as described in claim 5.

7. The recombinant strain according to claim 6, characterized in that: The recombinant strain is hosted by Escherichia coli and co-expresses inositol-1-phosphate synthase Ino1, methanol dehydrogenase Mdh, 3-hexose-6-phosphate synthase Hps, 6-phosphate-3-hexose isomerase Phi, endogenous phosphatase Suhb, and uronic acid dehydrogenase Udh.

8. The recombinant strain according to claim 7, characterized in that: The *E. coli* strain in question is *E. coli* JM109(DE3) with the *frmRAB*, *rpiA*, *pfkA*, and *pfkB* genes knocked out.

9. A method for producing D-gluconic acid, characterized in that: The recombinant strain according to any one of claims 6 to 8 is inoculated into a culture medium containing a carbon source for fermentation culture, and the expression of fused inositol oxygenase is induced to catalyze the production of D-gluconic acid from the substrate.

10. The use of the fusion inositol oxygenase according to any one of claims 1 to 3 in increasing the yield of D-gluconic acid.

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