Application of negative regulation XC4101 in promoting production of colorless xanthan gum from xanthomonas campestris pv. Campestris var. Campestris

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of negative regulation XC4101 in promoting production of colorless xanthan gum from xanthomonas campestris pv. Campestris. According to the invention, an Xcc 8004 genome is subjected to knockout modification, and a gene XC4101 in the Xcc 8004 genome is deleted, so that an Xcc 8004 mutant strain (mutant strain Xcc 4101) for producing colorless xanthan gum is obtained; the yield of colorless xanthan gum of the obtained mutant strain Xcc 4101 can reach 5.56 g / L, which is slightly higher than the yield (5.50 g / L) of xanthan gum produced by wild fungus Xcc 8004.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of negative regulation of XC_4101 in promoting the production of colorless xanthan gum by Xanthomonas brasiliensis pathogenic species of rapeseed. Background Technology

[0002] Xanthan gum is a natural extracellular polysaccharide produced by Xanthomonas species. It possesses excellent thickening, suspending, emulsifying, and stability properties, and is widely used in food processing, pharmaceuticals, oil extraction, and cosmetics manufacturing, among other fields. It is one of the world's largest-produced and most widely used microbial polysaccharides. As the world's largest producer of xanthan gum through industrial fermentation, my country accounts for approximately 70% of global production annually, holding a core position in the global xanthan gum industry. With economic development and the continuous expansion of downstream applications, market demand for xanthan gum is showing a rapid growth trend. How to further improve xanthan gum production efficiency and optimize product quality has become a key issue in promoting the upgrading of my country's xanthan gum industry.

[0003] Currently, industrial xanthan gum producing strains are mainly obtained through traditional mutagenesis and selection. This method suffers from problems such as high randomness in the mutagenesis process, long screening cycles, and high time and labor costs. Furthermore, the resulting strains often exhibit poor genetic stability and easily degraded gum-producing capacity, making it difficult to meet the high performance standards required for industrial production. In recent years, genetic engineering technology has provided new strategies for the targeted modification of Xanthan bacillus strains. By editing and regulating key genes in the strain's genome, precise optimization of traits such as xanthan gum yield and quality can be achieved. However, existing technologies for targeted improvement of key traits such as xanthan gum color regulation and synergistic yield enhancement still have shortcomings. How to obtain colorless and high-yielding xanthan gum producing strains remains a pressing technical challenge in this field.

[0004] Therefore, targeted modification of Xanthomonas aeruginosa through genetic engineering to discover key genes regulating xanthan gum synthesis and quality is an effective way to overcome existing technological bottlenecks and upgrade the xanthan gum industry. Research in this direction has significant theoretical and practical implications for promoting innovation in xanthan gum production technology and enhancing the core competitiveness of my country's xanthan gum industry. Summary of the Invention

[0005] Based on this, the present invention employs modern molecular biology techniques to target the pathogenic species of Xanthomonas spp. in rapeseed (…). Xanthomonas campestris pv. campestris 8004 Xcc 8004) was removed and modified. Xcc The gene XC_4101 in the 8004 genome was used to obtain a recombinant genetically engineered bacterium that produces colorless xanthan gum.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In a first aspect, the present invention provides a method for negatively regulating the XC_4101 gene in promoting the pathogenicity of Xanthomonas spp. in rapeseed ( Xanthomonas campestris pv. campestris The application of XC_4101 gene in colorless xanthan gum produced by 8004 includes any one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still having the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae pathogenic strain 8004; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

[0007] Preferably, in the above applications, the production of colorless xanthan gum is promoted by knocking out or knocking down the XC_4101 gene in Xanthomonas spp. 8004, a pathogenic strain of Xanthomonas spp.

[0008] Secondly, this invention provides a method for negatively regulating the XC_4101 gene in promoting the pathogenicity of Xanthomonas laurentii in *Rhizopus laurentii* species (…). Xanthomonas campestris pv. campestris The application of XC_4101 in colorless xanthan gum produced by 8004, wherein the nucleotide sequence of the XC_4101 gene includes any one of the following, and the amino acid sequence of the XC_4101 protein includes any one of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) The amino acid sequence shown in SEQ ID NO.2 is obtained by substitution, insertion or deletion of one or more amino acids, and still has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

[0009] Preferably, in the above applications, the production of colorless xanthan gum is promoted by knocking out or knocking down the XC_4101 gene in Xanthomonas spp. 8004, a pathogenic strain of Xanthomonas spp.

[0010] Thirdly, the present invention provides a mutant strain of *Xanthomonas spp.* var. *spp.* 8004, which is obtained by knocking out or down-knocking out the XC_4101 gene; the nucleotide sequence of the XC_4101 gene includes any one of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still promoting the production of colorless xanthan gum by Xanthomonas spp. 8004; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

[0011] Fourthly, the present invention provides a method for constructing the above-mentioned Xanthomonas spp. xanthophyllosis strain 8004 mutant strain, the method comprising: constructing a knockout plasmid based on the XC_4101 gene, transforming the knockout plasmid into Escherichia coli to obtain transformants, transferring the transformants into Xanthomonas spp. xanthophyllosis strain 8004, and then screening to obtain the Xanthomonas spp. xanthophyllosis strain 8004 mutant strain.

[0012] Fifthly, the present invention provides a method for producing colorless xanthan gum, the method comprising: fermenting and culturing the Xanthomonas oryzae mutant strain 8004 as described in claim 4 to obtain colorless xanthan gum.

[0013] Preferably, in the above method, the fermentation culture is performed by shake flask fermentation.

[0014] Preferably, in the above method, the fermentation culture temperature is 25℃-35℃.

[0015] Preferably, in the above method, the culture medium for fermentation is NYG liquid medium.

[0016] More preferably, in the above method, 2%-6% by mass of sucrose is added to the NYG liquid culture medium.

[0017] The beneficial effects of this invention include: This invention targets the pathogenic strain of Xanthomonas laurentii (a bacterium that causes disease in rapeseed)... Xanthomonas campestris pv. campestris 8004 Xcc 8004) was removed and modified. Xcc Gene XC_4101 in the 8004 genome is used to obtain colorless xanthan gum. Xcc 8004 mutant strain (mutant strain) Xcc △4101); the obtained mutant strain Xcc △4101's colorless xanthan gum yield can reach 5.56 g / L, slightly higher than that of wild mushrooms. Xcc The yield of xanthan gum produced by 8004 was 5.50 g / L. Attached Figure Description

[0018] Figure 1 wild strain Xcc PCR identification of the XC_4101 gene knockout strain in lane 8004; lane M represents the DNA marker DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); lane 1 represents the wild-type strain. Xcc 8004 amplified fragment; 2 is a mutant strain Xcc Amplified fragment of △4101; Figure 2 For qualitative analysis of mutant strains Xcc △4101 and wild mushrooms Xcc 8004's ability to synthesize flavin (colony diagram); Figure 3 mutant strain Xcc △4101 and wild mushrooms Xcc Comparison chart of xanthan gum production (8004); Figure 4 To increase the carbon source in the fermentation medium for mutant strains Xcc △Comparison chart of 4101 colorless raw gum production. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0022] In the following examples, the culture medium components used are as follows: LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L; NYG liquid medium: peptone 5 g / L, yeast extract 3 g / L, glycerol 20 g / L; 10% sucrose NYG liquid medium: peptone 5 g / L, yeast extract 3 g / L, glycerol 20 g / L, sucrose 100 g / L; In addition, when preparing the corresponding solid culture medium, 15 g / L of agar powder should be added; furthermore, when preparing the above-mentioned resistance culture medium, the working concentration of kanamycin (Km) is 30 µg / mL and the working concentration of rifampicin (Rif) is 30 µg / mL.

[0023] In the following examples, restriction endonucleases, DNA ligases, and other reagents were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; PCR primers were purchased from Sangon Biotech (Shanghai) Co., Ltd., Wuhan Branch; and wild-type strains of Xanthomonas spp. were used. Xcc 8004 originates from the South China Agricultural University Collection Center. It is a wild strain of *Xanthomonas brasiliensis*. Xcc 8004 is rifampicin resistant.

[0024] Example 1: Construction of gene knockout recombinant plasmid This embodiment discloses a method for constructing gene knockout recombinant plasmids, aiming to delete gene knockout using a two-stage homologous recombination method. Xcc The XC_4101 gene knockout recombinant plasmid was extracted from the 8004 genome to construct an engineered bacterium that produces colorless xanthan gum. The construction method of the XC_4101 gene knockout recombinant plasmid is as follows: (1) Design PCR primer pairs P1-F and P2-R for the upstream fragment (approximately 520 bp) of the XC_4101 gene, and PCR primer pairs P3-F and P4-R for the downstream fragment (approximately 530 bp) of the XC_4101 gene, respectively; (2) with Xcc Using total DNA from 8004 as a template, the upstream fragment of the XC_4101 gene was amplified using primers P1-F and P2-R, and the downstream fragment of the XC_4101 gene was amplified using primers P3-F and P4-R. (3) Using the fusion PCR method, the upstream and downstream fragments of the XC_4101 gene obtained in step S2 are used as templates, and P1-F and P4-R are used as primers to connect the upstream and downstream fragments of the XC_4101 gene to form the deletion fragment of the XC_4101 gene. (4) First, digest the XC_4101 gene deletion fragment obtained in step S3 with enzymes, and then ligate the digested fragment into pK18mobsacB. The recombinant plasmid that is correctly sequenced is the blocking plasmid pYMF-2 used for gene knockout.

[0025] In step (1), the sequences of P1-F, P2-R, P3-F and P4-R are shown in Table 1 below; Table 1 Sequences of P1-F, P2-R, P3-F, and P4-R

[0026] The PCR reaction system in step (2) is the same as in Table 1; the PCR amplification program is as follows: ① 95℃ pre-denaturation for 10 min; ② 95℃ denaturation for 30 s; ③ 56℃ annealing for 30 s; ④ 72℃ extension for 60 s; ⑤ Repeat ②~④ for 35 cycles; ⑥ 72℃ for 10 min. Store the PCR products at 4℃. The fusion PCR amplification program in step (3) is as follows: ① 95℃ pre-denaturation for 10 min; ② 95℃ denaturation for 30 s; ③ 56℃ annealing for 30 s; ④ 72℃ extension for 60 s; ⑤ Repeat ②~④ for 35 cycles; ⑥ 72℃ for 10 min; Store the PCR product at 4℃; The reaction system for fusion PCR is detailed in Table 2. Table 2 Fusion PCR reaction system in step (3) of Example 2

[0027] The enzyme used for digestion in step (4) is Bam HI and Hin d III was digested at 37℃ for 4 hours. The specific digestion reaction system is shown in Table 3 below.

[0028] Table 3 Enzyme digestion reaction system in step (4) of Example 2

[0029] The enzyme-digested fragments in step (4) were ligated into pK18mobsacB at a ligation temperature of 16℃ for 12 hours. The specific ligation system is shown in Table 4.

[0030] Table 4. The linkage reaction system in step (4) of Example 2

[0031] Example 2: Construction of engineered bacteria producing colorless xanthan gum This embodiment discloses a method for constructing a colorless xanthan gum-producing engineered bacterium, namely: constructing a colorless xanthan gum-producing engineered bacterium through a gene knockout method involving two homologous recombinations, including the following steps: (1) Competent Escherichia coli S17-1 was prepared using the CaCl2 induction method. The recombinant plasmid pYMF-2 from Example 2 was introduced into competent Escherichia coli S17-1 to obtain transformants. E . coliS17-1 / pYMF-2; (2) The transformant obtained in step (1) E . coli S17-1 / pYMF-2 is introduced via bonding transfer. Xcc 8004, then cultured on resistant plates to allow the XC_4101 gene deletion fragment on pYMF-2 to interact with... Xcc Homologous recombination exchange occurred in the XC_4101 gene fragment on the 8004 genome, and the single colony growing on the resistance plate was the first homologous recombination bacterium; (3) The first homologous recombinant strain obtained in step (2) is cultured for a second homologous recombination, and the second homologous recombinant strain is obtained by screening using the replication plate method; (4) Using primers P1-F and P4-R, the target gene fragment was amplified by PCR with the genome of the second homologous recombinant bacteria as a template; (5) Sequencing the amplified fragment from step (4) confirmed that gene XC_4101 had been deleted. The second homologous recombinant bacterium that successfully deleted gene XC_4101 is the genetically engineered recombinant bacterium that produces colorless xanthan gum. Xcc △4101 (mutant strain) Xcc △4101).

[0032] In step (1), the transformant is obtained. E . coli The specific method for S17-1 / pYMF-2 is as follows: ① Take 3 μL of pYMF-2 and add it to a volume of 100 μL. E . coli ① In S17-1 competent cells, incubate on ice for 30 min; ② After the ice bath, heat shock the system at 42℃ for 90 s; ③ Add 1 mL of antibiotic-free LB medium to the heat-shocked system and incubate at 37℃ for 1 h; ④ Spread the culture solution onto LB resistant plates containing Km and incubate at 37℃ for 10 h. The single colonies that grow on these plates are the S17-1 competent cells. E . coli S17-1 / pYMF-2 transformant; In step (2), the specific process of the joint transfer operation is as follows: ① Pick E . coli S17-1 / pYMF-2 was cultured in liquid LB medium at 37°C with shaking for 16 hours; ② Pick up Xcc8004 was cultured in NYG liquid medium at 30℃ with shaking for 24 h; ③ The bacterial suspensions from ① and ② were centrifuged and the bacterial cells were collected. The two bacterial cells were mixed in 1 mL of NYG liquid medium, washed twice, and resuspended in 0.1 mL of NYG liquid medium; ④ The bacterial resuspension from ③ was dropped onto antibiotic-free NYG plates and cultured upright for 48 h; In addition, since the first homologous recombinant bacteria have Km and Rif resistance, the first homologous recombinant bacteria were screened by adding Km and Rif to NYG plates. The plates were incubated upside down at 30℃ for 48 h, and the single colonies that grew were the first homologous recombinant bacteria; In step (3), the specific operation procedure for the second homologous recombination culture is as follows: ① The first homologous recombinant bacteria are placed in an antibiotic-free NYG liquid medium and cultured with shaking for 12-16 hours at a temperature of 30°C; ② The culture solution from step ① is spread on a 10% sucrose NYG plate containing Rif and cultured at 30°C for 48 hours to obtain single colonies; and the specific procedure for screening the second homologous recombinant bacteria is as follows: the single colonies obtained from the second homologous recombination culture are inoculated onto NYG plates containing Km and Rif and NYG plates containing only Rif, and the single colonies sensitive to Km are the second homologous recombinant bacteria; In step (4), the forward primer sequence P1-F and the reverse primer sequence P4-R were verified by PCR. The verification PCR reaction system is detailed in Table 5. The PCR reaction program was as follows: ① 95℃ pre-denaturation for 10 min; ② 95℃ denaturation for 1 min; ③ 56℃ annealing for 30 s; ④ 72℃ extension for 2 min; ⑤ Repeat ②~④ for 35 cycles; ⑥ 72℃ for 10 min. The PCR products were stored at 4℃. Figure 1 As shown, the amplified fragment size is approximately 1kb.

[0033] Table 5. Verification PCR reaction system in step (4) of Example 3

[0034] For step (5), the amplified fragment obtained by verification PCR in step (4) was sent to the Wuhan branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing to prove that XC_4101 had been deleted, thus obtaining the gene knockout mutant strain. Xcc △4101 refers to the engineered bacteria used to produce colorless xanthan gum.

[0035] Example 3 Qualitative Analysis Xcc △4101's ability to synthesize flavin This embodiment discloses a method for analyzing gene knockout mutant strains using a plate qualitative method. Xcc The flavin synthesis capacity of △4101 is compared with that of wild-type strains in this embodiment. Xcc 8004, the specific steps are as follows: S1, respectivelyXcc △4101 and Xcc 8004 was inoculated into NYG liquid medium and incubated at 30°C for 24 h; S2, take 2μL of each Xcc △4101 culture medium and Xcc Culture medium 8004 was inoculated onto NYG antibiotic-free plates and incubated at 30°C for 3 days. Comparison Xcc △4101 and Xcc The colony color of 8004.

[0036] like Figure 2 As shown, gene knockout mutant strain Xcc △4101 colonies formed on NYG antibiotic-free plates were white, while Xcc The colonies formed by 8004 on NYG antibiotic-free plates were yellow, qualitatively indicating a gene knockout mutant strain. Xcc The flavin synthesis pathway of △4101 is blocked, thus preventing the synthesis of flavin.

[0037] Example 4: Preparation of colorless xanthan gum This invention provides a process for preparing xanthan gum using a shake-flask fermentation method. Details are as follows: Select mutant strains Xcc △A single colony of 4101 was cultured in 5 mL of liquid NYG medium at 30°C with shaking for 36 h at a shaking speed of 180 rpm; the bacterial concentration was adjusted to OD0.05. 600 After reaching 1.0, 2% of the culture was transferred to liquid NYG medium containing 4% glucose and cultured at 30°C with shaking for 5 days at 180 rpm to obtain the fermentation broth. Four times the volume of pre-cooled anhydrous ethanol was added to the fermentation broth while stirring, and the mixture was allowed to stand overnight at 4°C. The flocculent precipitate was collected as xanthan gum, dried in a 60°C oven, and weighed. Three replicates were performed for each sample. The wild-type strain was also used... Xcc Xanthan gum was prepared from 8004 single colonies using the above method, dried in an oven at 60℃, and weighed; the sample was replicated in triplicate.

[0038] According to the literature "Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes andpolysaccharide in Xanthomonas campestris pathovar campestrisThe yield of yellow collagen was determined by the method described in (Tang JL, Liu YN, Barber CE, Dow JM, Wootton JC, Daniels MJ. 1991. Mol Gen Genet 226:409-417), and the results were analyzed using Prism software.

[0039] The results of the yellow collagen yield determination are shown in Table 6 and Figure 3 As shown, wild mushrooms Xcc The 8004 strain produced 5.50 g / L of xanthan gum, while the mutant strain... Xcc △4101 produced 5.56 g / L of xanthan gum, while the mutant strain... Xcc The colorless xanthan gum yield of △4101 is slightly higher than that of wild mushrooms. Xcc Xanthan gum production of 8004.

[0040] Table 6 Results of the yellow collagen yield test

[0041] Example 6: Culture in different culture media Select mutant strains Xcc △A single colony of 4101 was cultured in 5 mL of liquid NYG medium at 30°C with shaking for 36 h at a shaking speed of 180 rpm; the bacterial concentration was adjusted to OD0.05. 600 After reaching a concentration of 1.0, 2% of the xanthan gum was transferred to 50 mL of NYG culture medium and NYG culture medium with an increased sucrose content of 2%-6% for fermentation. The fermentation conditions were 30℃, 180 rpm, and a fermentation period of 5 days. The xanthan gum yield in different liquid culture media was then tested using the method described above. Details are shown in Table 7 below.

[0042] Table 7. Xanthan gum yield in different liquid culture media

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not 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. Negative regulation of the XC_4101 gene in promoting the pathogenicity of Xanthomonas brasiliensis in *Rosa rugosa* species (… Xanthomonascampestris pv. campestris The application of XC_4101 gene in colorless xanthan gum produced by 8004 includes any one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still having the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae pathogenic strain 8004; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

2. Negative regulation of the XC_4101 gene in promoting the pathogenicity of Xanthomonas brasiliensis in *Rhizopus spp.* (*Rhizopus brasiliensis*) Xanthomonascampestris pv. campestris The application of XC_4101 in colorless xanthan gum produced by 8004, wherein the nucleotide sequence of the XC_4101 gene includes any one of the following, and the amino acid sequence of the XC_4101 protein includes any one of the following: (1) The amino acid sequence as shown in SEQ ID NO.2; (2) The amino acid sequence shown in SEQ ID NO.2 is obtained by substitution, insertion or deletion of one or more amino acids, and still has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

3. The application according to claim 1 or 2, characterized in that, The goal of promoting the production of colorless xanthan gum was achieved by knocking out or knocking down the XC_4101 gene in Xanthomonas oryzae var. oryzae 8004, a pathogenic strain of Xanthomonas oryzae.

4. A mutant strain of Xanthomonas oryzae, pathogenic to wild rapeseed, named 8004, characterized by: The Xanthomonas oryzae pathogenic strain 8004 was obtained by knocking out or downsampling the XC_4101 gene; the nucleotide sequence of the XC_4101 gene includes any one of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence as shown in SEQ ID NO.1 obtained by substitution, deletion or insertion of one or more nucleotides, and still promoting the production of colorless xanthan gum by Xanthomonas spp. 8004; (3) A nucleotide sequence that is complementary to the nucleotide sequence described in (1) or (2), and can hybridize with it under strict conditions, and has the function of promoting the production of colorless xanthan gum by Xanthomonas oryzae 8004.

5. The method for constructing the Xanthomonas brasiliensis pathogenic strain 8004 mutant strain according to claim 4, characterized in that, The construction method includes: constructing a gene knockout plasmid based on the XC_4101 gene, transforming the knockout plasmid into Escherichia coli to obtain transformants, transferring the transformants into Xanthomonas spp. 8004, and then screening to obtain Xanthomonas spp. 8004 mutant strains.

6. A method for producing colorless xanthan gum, characterized in that, The method includes: fermenting and culturing the Xanthomonas oryzae mutant strain 8004 as described in claim 4 to obtain colorless xanthan gum.

7. The method according to claim 6, characterized in that, The fermentation culture method is shake-flask fermentation.

8. The method according to claim 6 or 7, characterized in that, The fermentation culture temperature is 25℃-35℃.

9. The method according to claim 6 or 7, characterized in that, The fermentation culture medium was NYG liquid medium.

10. The method according to claim 9, characterized in that, Add 2%-6% sucrose to NYG liquid medium.

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