Gluconobacter oxydans, gene target for improving l-sorbose synthesis, recombinant bacteria and application

Abstract

The application discloses an oxidizing gluconic acid bacillus, a gene target point for improving L-sorbose synthesis, a recombinant bacterium and application. The application obtains a high-yield mutant strain M-24 of L-sorbose through ARTP mutagenesis, and the mutant strain has good genetic stability. Transcriptomic analysis combined with gene function verification shows that overexpression of a glycosyltransferase gene gtrB or knockout of a ribose transport protein gene rbsB can significantly improve the L-sorbose production intensity of a wild-type strain under a high-concentration substrate condition, thereby providing a reference for directional improvement of an industrial strain.

Description

Technical Field

[0001] This invention belongs to the field of microbial mutagenesis breeding and biosynthesis technology, specifically involving a glucosamine oxidase, a gene target for enhancing L-sorbose synthesis, a recombinant bacterium, and its applications. Background Technology

[0002] L-sorbose is a naturally occurring hexose ketose, a representative industrial platform compound in the rare sugar family, possessing a sweetness and reducing properties similar to sucrose. As a core intermediate in the two-step fermentation process for vitamin C, L-sorbose is derived from D-sorbitol through bio-oxidation and is a key precursor for the industrial production of vitamin C. Recent studies have revealed that L-sorbose extends beyond traditional industrial applications, inducing apoptosis in cancer cells by interfering with glucose metabolism pathways, demonstrating its potential value in the biomedical field.

[0003] *Glucobacterium oxidans* is the main strain used in the industrial production of L-sorbose. Its cell membrane contains sorbitol dehydrogenase, which catalyzes the formation of L-sorbose from D-sorbitol. Industrial production typically employs high substrate concentrations to achieve high yields and high production intensity. However, under such high D-sorbitol conditions, wild-type *Glucobacterium oxidans* (… G. oxydans The cell activity and biotransformation efficiency of *Gluconobacterium oxidans* are significantly inhibited. Although fed-batch fermentation can partially alleviate substrate inhibition, it often leads to prolonged fermentation time and limited final yield. Therefore, improving the synthetic performance of *Gluconobacterium oxidans* at high D-sorbitol concentrations through genetic engineering or adaptive laboratory evolution is crucial for increasing L-sorbose yield.

[0004] While metabolic engineering can rationally overcome the performance bottlenecks of wild-type strains by reconstructing cellular metabolism and enhancing stress tolerance, the lack of gene editing tools and low homologous recombination efficiency severely restrict targeted modification of non-model strains like *Glucobacterium oxidans*. Against this backdrop, random mutagenesis combined with high-throughput screening has become a practical strategy for rapidly improving phenotypes. Its advantage lies in the simultaneous introduction of beneficial mutations at multiple sites, synergistically optimizing substrate tolerance and product synthesis capabilities. However, existing research largely focuses on screening high-yielding mutants, lacking in-depth analysis of molecular mechanisms. In recent years, multi-omics technologies have provided powerful tools for deciphering the evolutionary mechanisms of strains, potentially fundamentally solving the shortcomings of traditional strains in high-concentration substrate fermentation processes. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] One objective of this invention is to provide a high-yield L-sorbitol mutant strain of *Gluconobacterium oxysporum* M-24 obtained through ARTP mutagenesis.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a glucosidobacterium oxidans ( Gluconobacter oxydans The strain M-24 was deposited at the China Center for Type Culture Collection on March 30, 2026, with accession number CCTCC NO: M 2026559.

[0009] Preferably, the strain achieves an L-sorbose yield of not less than 4.0 g / L / h under shake-flask fermentation conditions using 300 g / L D-sorbitol as a substrate; Under fermentation conditions of 3 L fermenter and 300 g / L D-sorbitol as substrate, the yield of L-sorbose is not less than 10.0 g / L / h.

[0010] Another object of the present invention is to provide a gene target combination for improving the L-sorbose synthesis efficiency of *Glucosamine oxidans*, the gene target combination comprising overexpression of the gtrB gene and / or knockout of the rbsB gene; the nucleotide sequence of the gtrB gene is shown in SEQ ID NO.1, and the nucleotide sequence of the rbsB gene is shown in SEQ ID NO.5.

[0011] Another object of the present invention is to provide a recombinant glucosidobacterium oxidase that produces high levels of L-sorbose, wherein the recombinant bacteria uses glucosidobacterium oxidase as a host, overexpresses the gtrB gene, and / or knocks out the rbsB gene; the nucleotide sequence of the gtrB gene is shown in SEQ ID NO.1, and the nucleotide sequence of the rbsB gene is shown in SEQ ID NO.5.

[0012] Preferably, the host is *Glucosamine oxyphylla* ATCC 621.

[0013] Preferably, the recombinant bacteria also overexpress at least one gene selected from mdtA_2 and exoZ; and / or, the recombinant bacteria also knock out the eryI gene; The nucleotide sequence of the mdtA_2 gene is shown in SEQ ID NO.2, the nucleotide sequence of the exoZ gene is shown in SEQ ID NO.3, and the nucleotide sequence of the eryI gene is shown in SEQ ID NO.6.

[0014] Another object of the present invention is to provide the use of *Glucosobacterium oxidans* M-24 as described above in the production of L-sorbitol.

[0015] Another object of the present invention is to provide the use of the recombinant glucosidobacterium oxidans as described above in the production of L-sorbitol.

[0016] Preferably, the application involves preparing L-sorbose using D-sorbitol as a substrate through biotransformation of the *Glucosamine oxidans* M-24 or the recombinant *Glucosamine oxidans*.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention obtained a high-yielding L-sorbose mutant strain, M-24, through ARTP mutagenesis. Mutant strain M-24 reached its maximum conversion rate after 60 h of shake-flask fermentation, with a production intensity of 4.5 g / L / h, 3.2 times that of the wild-type strain. In scale-up culture in a 3 L fermenter, the production intensity further increased to 10.6 g / L / h. After 20 generations of subculturing, the growth and glycogen production performance of M-24 remained stable, exhibiting good genetic stability. Transcriptomic analysis combined with gene function verification showed overexpression of the glycosyltransferase gene. gtrB Or knock out the ribotransfer protein gene rbsB Both methods can significantly improve the L-sorbose production intensity of wild-type strains under high-concentration substrate conditions, providing a reference for the targeted improvement of industrial strains. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a graph showing the results of the ARTP mutagenic mortality rate determination in Example 1 of the present invention.

[0019] Figure 2 This is a diagram showing the standard curve plotted in Embodiment 1 of the present invention.

[0020] Figure 3 This is a diagram showing the initial screening results of the 96-well plate in Embodiment 1 of the present invention.

[0021] Figure 4The fermentation process curves of mutants M-24 and WT in a 3 L fermenter in Example 2 of this invention are shown.

[0022] Figure 5 This is a comparison chart of the growth curve and L-sorbose production intensity of mutant M-24 during the passage process in Example 3 of the present invention.

[0023] Figure 6 This is a differential gene volcano diagram in Example 4 of the present invention.

[0024] Figure 7 This is a diagram showing the verification results of gene overexpression and knockout in Example 7 of the present invention.

[0025] Figure 8 This is a diagram showing the fermentation results in Example 8 of the present invention. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0029] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0030] Glucosamine oxidase ( Gluconobacter oxydans The ATCC 621 sample was obtained from the China Center for Type Culture Collection (CCTCC). *E. coli* DH5α and plasmid pBBR1MCS-2 were from laboratory collections and publicly available. Homologous recombinases, restriction endonucleases, and In-Fusion PCR cloning kits were purchased from TaKaRa. All chemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd., and all biochemical reagents were analytical grade or higher.

[0031] Test methods used in the examples: Fermentation broth pretreatment: The fermentation broth was centrifuged at 15000 rpm for 10 min and the supernatant was collected. The supernatant sample was appropriately diluted and filtered through a 0.22 μm filter. The treated sample was then used for high performance liquid chromatography detection.

[0032] Determination of L-sorbose and D-sorbitol: High-performance liquid chromatography (HPLC) was used for detection. D-sorbitol and L-sorbose were quantitatively analyzed using a Bio-Rad Aminex HPX-87H column (maintained at 60°C) equipped with a refractive index detector, with 5 mM H₂SO₄ as the mobile phase and a flow rate of 0.4 mL / min. Compounds were identified by comparing retention times with standards.

[0033] The culture medium formulations involved in the following examples are as follows: LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L. When preparing solid medium, add 20 g / L agar powder to the above.

[0034] D-sorbitol medium: D-sorbitol 20 g / L, yeast extract 20 g / L. When preparing solid medium, add 20 g / L agar powder to the above.

[0035] D-sorbitol medium used for screening: D-sorbitol 300 g / L, yeast extract 20 g / L, solid medium with 2% agar.

[0036] Example 1: Mutagenesis and screening of high-yielding L-sorbitol strains ARTP mutagenesis: Inducing Glucosamine oxidans ( Gluconobacter oxydans ATCC 621 cells were cultured to the late exponential growth phase, collected by centrifugation, washed, and resuspended in sterile physiological saline to adjust cell density. 10 μL of the suspension was evenly spread onto a sterile glass slide and placed in the sample chamber of an ARTP mutagenesis system using high-purity helium as the working gas. Plasma treatment was performed at 120W radio frequency power, with the plasma nozzle maintaining a 2 mm gap from the sample surface. *Glucosamine oxidans* were exposed to ARTP for 0, 30, 60, 90, 120, 150, and 180 seconds, respectively. After treatment, the cells were appropriately diluted, spread on agar medium containing a high concentration of D-sorbitol, and incubated at 30°C for 48 hours. The optimal mutagenesis time was determined by calculating the lethality rate. Results are as follows: Figure 1 As shown.

[0037] Initial screening in 96-well plates: Mutagenic cells were seeded onto D-sorbitol agar plates and incubated at 30°C until single colonies formed. Single colonies were randomly selected and inoculated into 96-well plates, each containing 200 μL of culture medium (containing 200 g / L D-sorbitol). The plates were incubated at 30°C in a shaker (220 rpm). After the reaction, 50 μL of the culture supernatant was mixed with 50 μL of 3,5-dinitrosalicylic acid (DNS) reagent in a new 96-well plate. The plate was heated in a boiling water bath for 10 minutes. DNS was reduced to 3-amino-5-nitrosalicylic acid in the presence of reducing sugars, showing a reddish-brown color. After rapid cooling to room temperature, the absorbance was measured at 595 nm using a microplate reader. The concentration of L-sorbitol was quantified using a standard curve of known concentrations. The standard curve is shown below. Figure 2 As shown in the figure. Based on the initial screening, a total of 8 strains were selected: M-13, M-14, M-17, M-18, M-21, M-22, M-24, and M-27. The results are as follows. Figure 3 As shown.

[0038] Shake-flask secondary screening: The positive mutants M-13, M-14, M-17, M-18, M-21, M-22, M-24, and M-27 obtained from the initial screening were inoculated into D-sorbitol liquid medium and cultured overnight. Then, each candidate mutant was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of D-sorbitol medium (300 g / L D-sorbitol) and cultured at 30℃ with shaking at 220 rpm. The L-sorbose yield and final concentration were measured and compared with the wild-type strain. The results are shown in Table 1. Among them, mutant M-24 showed the highest yield, reaching 4.5 g / L / h, approximately 3.3 times that of the wild-type (1.4 g / L / h).

[0039] Table 1. Determination of L-sorbitol yield from rescreened strains.

[0040] Example 2: Fermentation test of high-yield L-sorbose strain M-24 L-sorbitol production was carried out in a 3-liter fermenter with a working volume of 1.0 L. The fermentation medium contained 300 g / L L-sorbitol and 20 g / L yeast extract. Fermentation was conducted at 30°C, pH maintained at 6.0, and a stirring speed of 700 rpm. An antifoaming agent was added as needed to inhibit foam formation. The biotransformation process was monitored for 4 days, with periodic sampling and analysis of cell growth, substrate consumption, and product formation.

[0041] Test results are as follows Figure 4 As shown. WT requires approximately 40 hours to reach peak conversion, with a production intensity of 7.2 g / L / h. Figure 4a). In contrast, M-24 achieves maximum conversion in just 27 hours, with a production intensity of 10.6 g / L / h ( Figure 4 (b) It exhibits significant advantages in rate and efficiency under industrial-related conditions. The higher production intensity observed in the fermenter is attributed to improved process control and oxygen transfer, further supporting the industrial potential of M-24.

[0042] Example 3: Genetic stability of high-yielding L-sorbose strain M-24 The M-24 mutant strain was streaked onto D-sorbitol agar plates and cultured for 20 generations, with a single colony randomly selected from each generation. Colonies from generations 1, 5, 10, and 20 were then inoculated into D-sorbitol medium and fermented in shake flasks under the same conditions. Genetic stability was assessed by comparing the L-sorbitol growth intensity of each generation. Results are as follows: Figure 5 As shown, by Figure 5 As shown by the growth curves, the growth curves of the 1st, 5th, 10th, and 20th generations of the strain basically overlapped, and there was no significant difference in the time and level at which the cell density reached its peak. Figure 5 As shown in Table 2, the L-sorbitol production intensity of each generation of strains remained stable, with no significant performance decline. Specific production intensity data are shown in Table 2.

[0043] Table 2 Genetic stability of Glucosamine oxidase M-24

[0044] Example 4: Transcriptomics sequencing analysis of gene loci that may promote L-sorbose synthesis To investigate the transcriptional mechanism of enhanced L-sorbose production in the mutant, transcriptomic analysis was performed on wild-type and mutant strains. The strains were cultured in D-sorbitol medium to the exponential growth phase, and cells were collected. The culture medium was centrifuged (5000 r / min, 5 min, 4℃) to collect the cell pellet, washed twice with phosphate-buffered saline (PBS, pH 7.4), then flash-frozen in liquid nitrogen and stored at -80℃ for later use. RNA extraction, library construction, and sequencing were performed by Genewiz (Suzhou, China) using the Illumina sequencing platform.

[0045] Transcriptomics sequencing results are shown in Figure 6Cells from both wild-type and M-24 strains were collected during the logarithmic fermentation phase. Based on strict threshold criteria (|log2(fold change)|>2 and qvalue≤0.05), 21 significantly differentially expressed genes (DEGs) were identified, including 3 upregulated genes and 18 downregulated genes. Functional annotations of the differentially expressed genes are shown in Table 3. According to the KEGG functional annotation results of the differentially expressed genes, most genes are difficult to directly link to L-sorbate synthesis-related pathways. Among them, the gene encoding polyisoprene phosphate glycosyltransferase, which participates in cell membrane glycosylation, is... gtrB The gene was significantly upregulated. This enhancement may be achieved by improving membrane integrity and homeostasis, which has significant advantages for high substrate fermentation environments.

[0046] Transcriptome analysis of strain M-24 revealed a synergistic downregulation of genes involved in alternative carbon source uptake and catabolism. Notably, the ribotransporter operon (… rbsABC The activity was significantly suppressed. This pattern suggests a metabolic strategy of increasing the availability of resources required for the target biotransformation by diverting carbon flux from competing pathways.

[0047] Table 3. Differentially expressed genes and functional annotations in strain M-24

[0048] Example 5: Construction of gene overexpression plasmids The broadly host-range plasmid pBBR1MCS-2 was used as the expression vector. The pBBR1MCS-2 backbone was linearized by PCR, and the sequence of the gene to be overexpressed was amplified from wild-type genomic DNA using primers carrying homologous arms. The linearized vector and gene were assembled via homologous recombination and transformed into competent *E. coli* DH5α cells. Transformants were screened on LB agar supplemented with kanamycin (50 μg / mL) and confirmed by colony PCR. The recombinant plasmid was then sequenced. The confirmed recombinant plasmid and empty vector were introduced into competent *Gluconobacter oxidans* cells via electroporation.

[0049] (1) Construction of recombinant strain WT-1 The overexpression plasmid pBBR1MCS-2- was constructed using the method described above. gtrB The recombinant plasmid was then sequenced. The confirmed recombinant plasmid and empty vector were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, overexpressing plasmids were obtained. gtrB The recombinant strain WT-1. The nucleotide sequence of the gtrB gene is shown in SEQ ID NO.1.

[0050] (2) Construction of recombinant strain WT-2 The overexpression plasmid pBBR1MCS-2- was constructed using the method described above. mdtA_2 The recombinant plasmid was then sequenced. The confirmed recombinant plasmid and empty vector were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, overexpressing plasmids were obtained. mdtA_2 The recombinant strain WT-2 has the mdtA_2 gene. The nucleotide sequence of the mdtA_2 gene is shown in SEQ ID NO.2.

[0051] (3) Construction of recombinant strain WT-3 The overexpression plasmid pBBR1MCS-2- was constructed according to the method in Example 5. exoZ The recombinant plasmid was then sequenced. The confirmed recombinant plasmid and empty vector were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, overexpressing plasmids were obtained. exoZ The recombinant strain WT-3 contains the exoZ gene. The nucleotide sequence of the exoZ gene is shown in SEQ ID NO.3.

[0052] Example 6: Construction of gene knockout frames A knockout mutant was constructed by replacing the target gene with a kanamycin resistance cassette (KanR) using homologous recombination technology. Using gene-specific primers, approximately 800 bp upstream and downstream homologous arms (DHA) of the gene to be knocked out were amplified from wild-type genomic DNA. The KanR cassette was obtained from the pBBR1MCS-2 vector. After assembling these three fragments (UHA–KanR–DHA) into a knockout cassette, the successfully validated knockout cassette was introduced into competent *Glucophage oxidans* cells via electroporation. Transformants were screened on D-sorbitol agar plates containing kanamycin, and the correct gene substitution was confirmed by colony PCR.

[0053] (1) Construction of recombinant strain WT-4 Construct according to the above method sufS_2 Gene knockout frames were then used, and successfully validated knockout frames were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, [the desired results were obtained]. sufS_2 The gene-knockout recombinant bacterium WT-4. The nucleotide sequence of the sufS_2 gene is shown in SEQ ID NO.4.

[0054] (2) Construction of recombinant strain WT-5 Construct according to the above method rbsB Gene knockout frames were then used, and successfully validated knockout frames were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, [the desired results were obtained]. rbsB The gene knockout recombinant bacterium WT-5. The nucleotide sequence of the rbsB gene is shown in SEQ ID NO.5.

[0055] (3) Construction of recombinant strain WT-6 Construct according to the above method eryI Gene knockout frames were then used, and successfully validated knockout frames were introduced into competent *Glucosamine oxidans* cells via electroporation. After screening, [the desired results were obtained]. eryI The gene-knockout recombinant bacterium WT-6. The nucleotide sequence of the eryI gene is shown in SEQ ID NO.6.

[0056] Example 7: Validation of overexpression and knockout strains The results of overexpression and knockout strain validation are shown in [link to documentation]. Figure 7 For the overexpressing strains, we extracted total RNA from each recombinant strain and the wild-type control, and after reverse transcription, we used real-time quantitative PCR to detect the transcription level of the target gene. Figure 7 As shown in (a), (b) and (c), the expression levels of the corresponding genes in the three overexpression strains were significantly upregulated compared with the wild-type strain, confirming the effective expression of the overexpression vector.

[0057] For the knockout strains, we used colony PCR for verification. The deletion of the sufS_2 gene resulted in the strain's inability to grow, and no transformants were obtained. The lengths of the rbsB, eryI, and kanamycin resistance gene cassette fragments were 1042 bp, 625 bp, and 895 bp, respectively. Figure 7 As shown in (d), agarose gel electrophoresis showed that all knockout strains amplified specific bands of the expected size, confirming the successful replacement of the target gene.

[0058] Example 8: Fermentation production of L-sorbose by recombinant strain and control strain The prepared recombinant strains WT-1, WT-2, WT-3, WT-4, WT-5 and the control strain WT were selected and activated on D-sorbitol medium. The activated seed cultures were then inoculated into shake flasks containing D-sorbitol 300 g / L and yeast extract 20 g / L, and fermented at 30℃ and 220 rpm. The fermentation results are as follows. Figure 8 As shown in Table 4.

[0059] Depend on Figure 8 From a, we can know that gtrB Overexpression of the compound significantly promoted the growth of the strain, shortening the lag phase and increasing the final biomass, confirming its positive role in L-sorbose biotransformation. Overexpression... mdtA_2 and exoZ The growth of the strain was significantly inhibited, and its OD 600 The value remained consistently low throughout the entire culture period. Figure 8 b indicates that WT-gtrB exhibits significantly higher L-sorbose production intensity than the wild type, while overexpression... mdtA_2 or exoZ The strain produced almost no L-sorbose.

[0060] Depend on Figure 8 c indicates that knockout rbsB The growth rate of this strain is superior to that of the wild type. Knockout eryI The growth of strain (ΔeryI) was severely inhibited, and its OD... 600 The value remained consistently low throughout the entire culture period. Figure 8 As shown in d, the L-sorbose production intensity of ΔrbsB was significantly higher than that of the wild type, while the L-sorbose yield of ΔeryI was extremely low. The results indicate that... rbsB The absence of this component helps alleviate the inhibition of strain growth by high substrate concentrations, thereby increasing L-sorbose production intensity. eryI It is essential for maintaining normal growth and product synthesis of the strain; its absence leads to severe growth defects and yield loss.

[0061] As shown in Table 4, compared with the control strain WT, the yield of WT-1 increased by 50% and that of WT-4 increased by 150%.

[0062] Table 4 Fermentation Results

[0063] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0064] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A type of glucosinolate bacillus ( Gluconobacter oxydans M-24, characterized in that, The strain was deposited at the China Center for Type Culture Collection on March 30, 2026, with accession number CCTCC NO: M 2026559.

2. The *Gluconobacterium oxidans* M-24 as described in claim 1, characterized in that, Under shake-flask fermentation conditions with 300 g / L D-sorbitol as substrate, the strain yielded an L-sorbose yield of not less than 4.0 g / L / h. Under fermentation conditions of 3 L fermenter and 300 g / L D-sorbitol as substrate, the yield of L-sorbose is not less than 10.0 g / L / h.

3. A gene target for enhancing L-sorbose synthesis in *Glucosamine oxidans*, characterized in that, The gene targets include overexpression of the gtrB gene and / or knockout of the rbsB gene; the nucleotide sequence of the gtrB gene is shown in SEQ ID NO.1, and the nucleotide sequence of the rbsB gene is shown in SEQ ID NO.

5.

4. A recombinant glucosidobacterium oxidase that produces high levels of L-sorbose, characterized in that, Using *Glucosamine oxidans* as a host, the gtrB gene is overexpressed and / or the rbsB gene is knocked out; the nucleotide sequence of the gtrB gene is shown in SEQ ID NO.1, and the nucleotide sequence of the rbsB gene is shown in SEQ ID NO.

5.

5. The recombinant glucosamine oxidase according to claim 4, characterized in that, The host is *Glucosamine oxyphylla* ATCC 621.

6. The recombinant glucosamine oxidase according to claim 4, characterized in that, The recombinant glucosinolate bacteria also overexpress at least one gene selected from mdtA_2 and exoZ; and / or, the recombinant bacteria also knock out the eryI gene.

7. The recombinant glucosamine oxidase according to claim 6, characterized in that, The nucleotide sequence of the mdtA_2 gene is shown in SEQ ID NO.2, the nucleotide sequence of the exoZ gene is shown in SEQ ID NO.3, and the nucleotide sequence of the eryI gene is shown in SEQ ID NO.

6.

8. The use of Gluconobacterium oxidans M-24 as described in claim 1 in the production of L-sorbitol.

9. The application of the recombinant glucosidobacterium oxidans as described in claim 4 in the production of L-sorbitol.

10. The application according to claim 8 or 9, characterized in that, The application involves preparing L-sorbose through biotransformation using D-sorbitol as a substrate.

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