Application of negative regulation XC1141 gene in improving yield of xanthan gum produced by xanthomonas campestris pv. Campestris var. Campestris

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to application of a negative regulation XC1141 gene in improving the yield of xanthan gum produced by xanthomonas campestris var. Campestris. According to the invention, an Xcc 8004 genome is subjected to knockout modification, and a gene XC1141 in the Xcc 8004 genome is deleted, so that an Xcc 8004 mutant strain (mutant strain Xcc 1141) with high yield of xanthan gum is obtained; the xanthan gum yield of the obtained Xcc 8004 mutant strain can reach 9.11 g / L, and the yield is increased by 65% compared with that of wild fungus Xcc 8004 (5.52 g / L).

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of negative regulation of the XC_1141 gene in increasing the xanthan gum production of Xanthomonas spp. in pathogenic species of rapeseed. Background Technology

[0002] Xanthan gum is a natural extracellular polysaccharide secreted by Xanthomonas species. It possesses multiple functions, including thickening, stabilizing, and emulsifying, and is widely used in food processing, oil extraction, and pharmaceutical preparations. It is one of the most economically valuable products in the global biotechnology industry. China, as the core country for global xanthan gum industrial fermentation production, accounts for over 70% of global production annually, holding a dominant position in the international supply chain. With the continuous expansion of downstream applications, market demand for xanthan gum continues to rise. Improving its industrial production efficiency and stability has become a core issue supporting the sustainable development of my country's xanthan gum industry.

[0003] Currently, industrial xanthan gum production strains are mainly obtained through traditional mutagenesis and selection methods. This technology relies on random mutation and large-scale screening, which suffers from drawbacks such as high randomness, long cycles, and high screening costs. Furthermore, the selected strains are prone to poor genetic stability and degraded gum-producing capacity, making them unsuitable for the high-efficiency and stable strains required by modern industrial production. In recent years, genetic engineering technology has provided a new, precisely targeted approach for microbial strain improvement. However, research on the key gene networks and functions regulating xanthan gum biosynthesis in Xanthomonas remains insufficient, and there is a lack of precise modification strategies that can be directly applied to industrial production.

[0004] Faced with the bottlenecks of traditional technologies and the growth of market demand, the xanthan gum industry urgently needs more efficient and precise strain improvement technologies to overcome the limitations of current production efficiency. Analyzing the molecular regulatory mechanisms of xanthan gum synthesis and developing targeted gene modification technologies is a key direction for achieving a dual improvement in xanthan gum yield and quality. This technological breakthrough will not only solve the drawbacks of traditional mutagenesis breeding but also promote the transformation of my country's xanthan gum industry towards intelligent and precise production, consolidating its competitive advantage in the global market. 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_1141 in the 8004 genome was used to obtain a recombinant genetically engineered bacterium that produces high levels of xanthan gum.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: In a first aspect, embodiments of the present invention provide a method for negatively regulating the XC_1141 gene in enhancing the pathogenicity of Xanthomonas spp. in *Xanthomonas spp.* (a strain of *Xanthomonas spp.*). Xanthomonas campestris pv. campestris The application of Xanthan gum production in 8004, wherein the nucleotide sequence of the XC_1141 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 having the function of increasing the xanthan gum production of 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 increasing the xanthan gum production of Xanthomonas spp. 8004.

[0007] Preferably, in the above applications, the XC_1141 gene in Xanthomonas spp. 8004, a pathogenic strain of Xanthomonas spp., is knocked out or knocked down to increase the yield of xanthan gum.

[0008] Secondly, the present invention provides an application of negative regulation of the XC_1141 protein in increasing the xanthan gum production of Xanthomonas spp. pathogenic strains of rapeseed, wherein the amino acid sequence of the XC_1141 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 increasing the xanthan gum production of Xanthomonas spp. 8004.

[0009] Preferably, in the above applications, the XC_1141 gene in Xanthomonas spp. 8004, a pathogenic strain of Xanthomonas spp., is knocked out or knocked down to increase the yield of xanthan gum.

[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_1141 gene; the nucleotide sequence of the XC_1141 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 having the function of increasing the xanthan gum production of 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 increasing the xanthan gum production of Xanthomonas spp. 8004.

[0011] Fourthly, the present invention provides a method for constructing the above-mentioned Xanthomonas spp. xanthophyllosis strain 8004 mutant strain, the method comprising: obtaining a recombinant plasmid by constructing an expression plasmid of the XC_1141 gene; transforming the recombinant plasmid into Escherichia coli to obtain a transformant; transferring the transformant 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 increasing the xanthan gum production of Xanthomonas brasiliensis var. brasiliensis 8004, the method comprising: fermenting and culturing the Xanthomonas brasiliensis var. brasiliensis 8004 mutant strain as described in claim 4 to obtain 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] The beneficial effects of this invention include at least the following: This invention provides protection against *Xanthomonas laurentii* pathogenic strains of *Xanthomonas laurentii* (…). Xanthomonas campestris pv. campestris 8004 Xcc 8004) was removed and modified. Xcc The gene XC_1141 in the 8004 genome is used to obtain high xanthan gum production. Xcc The 8004 mutant strain (mutant Xcc△1141) yielded xanthan gum of 9.11 g / L, which was lower than that of the wild-type strain. Xcc The concentration of 8004 (5.52 g / L) increased by 65%. Attached Figure Description

[0016] Figure 1 The PCR validation results of the gene knockout plasmid pK18-XC_1141 are shown; lane M is the DNA marker DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp); lanes 1 and 2 are amplified fragments using the gene knockout plasmid pK18-XC_1141 as a template. Figure 2 wild strain XccPCR identification of XC_1141 gene knockout strains in lane 8004; lane M is the DNA marker DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); lanes 1 and 2 are wild-type strains. Xcc 8004 is an amplified fragment; 3 is an amplified fragment of the mutant strain Xcc△1141; Figure 3 The mutant strain Xcc△1141 and the wild-type fungus Xcc Comparison chart of xanthan gum production (8004); Xcc 8004 is a wild mushroom. Xcc 8004; Xcc△1141 is the XC_1141 mutant strain; This indicates a highly significant difference. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In the following examples, Xanthomonas spp. var. spp. (var. spp.) is a pathogenic strain of Xanthomonas spp. var. spp. (var. spp.) Xanthomonas campestris pv. campestris (abbreviated as) Xcc 8004; refers to *Xanthomonas spp.*, a pathogenic strain of *Xanthomonas spp.* in *Rosa spp.*, which is a wild-type fungus. Xcc 8004 was a gift from Professor Wang Haihong of South China Agricultural University.

[0021] In the following example, the engineered strain of Xanthomonas spp. var. spp. var. spp. is abbreviated as Xcc△1141; that is, the engineered strain of Xanthomonas spp. var. spp. is a mutant strain Xcc△1141.

[0022] In the following examples, unless otherwise specified, the experimental methods are all conventional experimental methods. Unless otherwise specified, the reagents and consumables used in the following examples are all commercially available products. All percentages (%) in the text represent mass fractions.

[0023] In the following examples, wild mushrooms Xcc 8004, Escherichia coli S17-1, and Xcc△1141 were deposited at the Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences; restriction endonucleases, ligases, and other reagents were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0024] Example 1 This invention provides an engineered strain of *Xanthomonas aeruginosa*, a pathogenic bacterium in *Rosa rugosa*, and its construction process. Details are as follows: (1) Design primers Primers were designed based on the XC_1141 gene (sequence shown in SEQ ID NO.1) and its upstream and downstream sequences (underlined parts are restriction enzyme sites), as shown in Table 1 below.

[0025] Table 1 Primers designed based on the XC_1141 gene and its upstream and downstream sequences.

[0026] (2) PCR amplification of gene fragments (2-1) First round of amplification by Xcc Using 8004 total DNA as a template, primers 1141-P1 / 1141-P2 were used to amplify the approximately 500bp upstream sequence P1P2 of XC_1141; primers 1141-P3 / 1141-P4 were used to amplify the approximately 500bp downstream sequence P3P4 of XC_1141. The amplification system was as follows: 50 μL: 1 μL total DNA, 1 μL each of primers 1141-P1 / 1141-P2 or 1 μL each of primers 1141-P3 / 1141-P4, and 47 μL high-fidelity PCR mix. The amplification conditions were as follows: pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, and extension at 72℃ for 15 s.

[0027] (2-2) Second round of amplification Using the first-round PCR product (a mixture of P1P2 and P3P4 fragments) as a template, the upstream and downstream fragments P1P4 of XC_1141 were amplified using 1141-P1 / 1141-P4 as primer pairs. The amplification system was as follows: 50 μL: 1 μL each of P1P2 and P3P4 fragments, 1 μL each of 1141-P1 / 1141-P4 primers, and 46 μL of high-fidelity PCR mix. The amplification conditions were as follows: pre-denaturation at 95℃ for 1 min, denaturation at 95℃ for 20 s, annealing at 56℃ for 20 s, and extension at 72℃ for 20 s.

[0028] (3) Constructing plasmids P1P4 fragment Bam HI and Hin After digestion with dIII, the fragment was ligated into pK18mobsacB to obtain plasmid pYMF-1. The digestion conditions were as follows: 60 μL of fragment or plasmid, 10 μL of 10× digestion buffer. Bam HI 2 μL, Hin Add 2 μL of d III to a final volume of 100 μL with double-distilled water and incubate at 37°C for 4 h. The ligation conditions are as follows: 10 μL of the digested fragment, 5 μL of the digested vector, 2 μL of 10× ligase buffer, 1 μL of DNA ligase, and double-distilled water to a final volume of 20 μL. Ligate at 16°C for 12 h.

[0029] (4) Transformation Escherichia coli (E. coli) was prepared using the CaCl2 induction method. E . coli S17-1 competent cells, add 3 μL of plasmid pYMF-1 to 100 μL E . coli After S17-1 competent cells were incubated on ice for 30 min, then heat-shocked at 42℃ for 90 s. 1 mL of antibiotic-free LB medium (10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride) was added. After culturing at 37℃ for 1 h, the cells were plated onto LB agar plates containing kanamycin (Km, 30 μg / mL) (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder). The plates were then incubated at 37℃ for 24 h to obtain transformants. E . coli S17-1 / pYMF-1.

[0030] (5) Joining transfer The blocking plasmid pYMF-1 was introduced into the wild-type strain using conjugation transfer. Xcc 8004. The specific operating steps are as follows: Select the transformant. E . coliA single colony of S17-1 / pYMF-1 was cultured overnight at 37°C with shaking in 5 mL of liquid LB medium; pick... Xcc Single colony 8004 was cultured overnight at 30°C with shaking in NYG liquid medium (5 g / L peptone, 3 g / L yeast extract, and 20 g / L glycerol). The two bacterial cells were collected by centrifugation, mixed, washed twice in 1 mL of NYG liquid medium, and resuspended in 0.1 mL of NYG liquid medium to obtain a bacterial suspension. The bacterial suspension was added dropwise to antibiotic-free NYG solid medium (5 g / L peptone, 3 g / L yeast extract, 20 g / L glycerol, and 15 g / L agar powder), air-dried, and incubated upright for 48 h. After incubation, the bacterial growth was washed with 1 mL of sterile water, diluted 100 times, and spread on NYG plates containing kanamycin (Km, 30 μg / mL) and rifampin (Rif, 50 μg / mL). The plates were incubated upside down at 30°C for 48 h to obtain a single colony of recombinant strain.

[0031] (6) Secondary reorganization The obtained recombinant strain was cultured overnight in NYG liquid medium at 30°C with shaking. It was then plated on NYG plates containing rifampicin (Rif, 50 μg / mL) and 10% sucrose and cultured at 30°C for 48 h to obtain single colonies. These single colonies were then inoculated onto NYG plates containing kanamycin (Km, 30 μg / mL), rifampicin (Rif, 50 μg / mL), and rifampicin (Rif, 50 μg / mL) (replication plate method) to screen for kanamycin-sensitive colonies.

[0032] (7) Validation of mutant strains PCR was used to amplify and screen kanamycin-sensitive colonies to obtain amplified fragments (i.e., the amplified fragment of the mutant strain Xcc△1141). The amplification system was as follows: 20 μL: 1 μL total DNA from kanamycin-sensitive bacteria, 1 μL each of primers 1141-P1 and 1141-P4, 10 μL of 2×PCR mix, and double-distilled water to bring the volume to 20 μL. The amplification conditions were as follows: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 56℃ annealing for 20 s, and 72℃ extension for 30 s.

[0033] wild mushrooms Xcc Wild-type fungi were obtained by amplification using 8004. Xcc 8004 amplification fragment, respectively wild fungus Xcc The amplified fragments of strain 8004 and mutant strain Xcc△1141 were subjected to gel electrophoresis. The test results are as follows: Figure 1 As shown, the results indicate that wild mushrooms XccThe 8004 amplified fragment is approximately 2.3 Kb in length, while the amplified fragment of the mutant strain Xcc△1141 is approximately 1.1 Kb in length, proving that the XC_1141 gene was deleted and the gene knockout mutant strain Xcc△1141 was successfully constructed.

[0034] In addition, the amplified fragment was sent to the Changsha branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the results were as follows. Figure 2 As shown, this further confirms that the XC_1141 gene was successfully knocked out.

[0035] Example 2 This invention provides a process for preparing xanthan gum using a shake-flask fermentation method. Details are as follows: Single colonies of the mutant strain Xcc△1141 were picked and placed in 5 mL of liquid NYG medium. The culture was incubated 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 a shaking speed of 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. Three parallel replicates of the sample were performed.

[0036] 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 campestris The 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.

[0037] The results of the yellow collagen yield determination are shown in Table 2 and Figure 3 As shown, wild mushrooms XccXanthan gum production was 5.52 g / L for strain 8004, while the xanthan gum production of mutant strain Xcc△1141 was 9.11 g / L, which was lower than that of the wild-type strain. Xcc The 8004 improved by 65%; statistical analysis results from Prism software showed that the difference between the two was extremely significant.

[0038] Table 2 Results of the yellow collagen yield test

[0039] 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_1141 gene in increasing the pathogenicity of Xanthomonas spp. in rapeseed ( Xanthomonascampestris pv. campestris The application of Xanthan gum production in 8004, wherein the nucleotide sequence of the XC_1141 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 having the function of increasing the xanthan gum production of 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 increasing the xanthan gum production of Xanthomonas spp. 8004.

2. Application of negative regulation of XC_1141 protein in increasing xanthan gum production in pathogenic species of *Xanthomonas laurentii*, wherein the amino acid sequence of the XC_1141 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 increasing the xanthan gum production of Xanthomonas spp. 8004.

3. The application according to claim 1 or 2, characterized in that, The goal of increasing xanthan gum production was achieved by knocking out or de-knocking the XC_1141 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 knocking down the XC_1141 gene; the nucleotide sequence of the XC_1141 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 having the function of increasing the xanthan gum production of 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 increasing the xanthan gum production of Xanthomonas spp. 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 an expression plasmid of the XC_1141 gene to obtain a recombinant plasmid, transforming the recombinant plasmid into Escherichia coli to obtain a transformant, transferring the transformant into Xanthomonas spp. 8004, and then screening to obtain Xanthomonas spp. 8004 mutant strains.

6. A method for increasing the xanthan gum yield of Xanthomonas brasiliensis var. brasiliensis 8004, characterized in that, The method includes: fermenting and culturing the Xanthomonas oryzae mutant strain 8004 as described in claim 4 to obtain 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℃.

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