Xanthan gum yield-related gene and application thereof
By introducing the xanthan gum production-related gene XC_2876 into Xanthomonas brasiliensis, an engineered strain was constructed, which solved the problem of insufficient xanthan gum production, significantly increased xanthan gum production, and enhanced its application effect in multiple fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGDONG FOOD & DRUG VOCATIONAL COLLEGE
- Filing Date
- 2023-04-26
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies are insufficient to effectively increase xanthan gum production, thus affecting its application in pharmaceuticals, food, beverages, cosmetics, detergents, and oil extraction.
By introducing the xanthan gum production-related gene XC_2876 into the genome of Xanthomonas oryzae, an engineered strain was constructed to optimize the xanthan gum production pathway and increase xanthan gum yield.
It significantly increased the yield of xanthan gum and enhanced its application potential in pharmaceuticals, food, beverages, cosmetics, detergents and oil extraction.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, and in particular to a gene related to xanthan gum production and its application. Background Technology
[0002] Xanthan gum is a pathogenic species of Xanthomonas spp. in rapeseed. Xanthomonas campestris pv. campestris , Xcc Xanthan gum, an extracellular polysaccharide polymer produced by microorganisms, is the most widely used of the developed microbial polysaccharides. Due to its excellent thickening and thixotropic properties, high stability to heat, acids, and alkalis, and compatibility with various solvents, xanthan gum can be used as a suspending agent, thickener, emulsifier, and stabilizer in pharmaceuticals, food, beverages, cosmetics, detergents, and oil extraction. Therefore, xanthan gum is a very important biotechnology product.
[0003] With the rapid development of molecular biology techniques and the whole-genome sequencing of xanthan gum-producing strains, the biosynthetic pathway of xanthan gum has become largely clear. Glucose-6-phosphate and fructose-6-phosphate produced intracellularly in *Xanthomonas* cells generate precursors for xanthan gum synthesis (GDP-mannose, UDP-glucose, UDP-glucuronic acid, etc.) through enzymatic reactions. The resulting pentose repeating units bind to intracellular membrane lipid carriers, are transported to the extracellular space, polymerize to form xanthan gum, and are then transported extracellularly via porins. The enzymes catalyzing these steps are all derived from… gum The gene cluster (approximately 16 kb) encodes, containing gumB ~ gumK A total of 12 genes form an operon, and the normal synthesis of xanthan gum requires the participation of the products encoded by all 12 genes. gumD , gumM , gumH , gumK , gumI They respectively encode glycosyltransferases, gumL Encoding a ketotransferase that acylates mannose pyruvate. gumF , gumG The genes encoding acetyltransferases, GumJ, GumB, GumC, and GumE, are involved in the polymerization and transport of xanthan gum. Xanthan gum synthesis in Xanthomonas also involves other basal metabolic processes, particularly sugar metabolism-related pathways, which influence xanthan gum production. Therefore, genetically modifying genes related to xanthan gum synthesis on the Xcc chromosome to control key enzyme systems in the xanthan gum synthesis metabolic pathway within the bacteria, thereby altering metabolic flux, is an important method to increase xanthan gum production. Summary of the Invention
[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a xanthan gum production-related gene and its application.
[0005] The objective of this invention is achieved through the following technical solution: a xanthan gum production-related gene, wherein the amino acid sequence encoded by the gene is shown in SEQ ID NO.1.
[0006] The nucleotide sequence of the xanthan gum production-related gene is shown in SEQ ID NO.2.
[0007] This invention also provides the application of the above-mentioned xanthan gum yield-related genes in xanthan gum production.
[0008] This invention also provides the application of the above-mentioned xanthan gum production-related genes in the construction of xanthan gum-producing engineered bacteria.
[0009] This invention also provides an engineered bacterium that produces xanthan gum, specifically a strain that expresses the protein encoded by the aforementioned xanthan gum production-related gene in the genome of Xanthomonas oryzae.
[0010] The present invention also provides a method for constructing the above-mentioned engineered bacteria that produce xanthan gum, comprising the following steps: introducing the nucleic acid molecule of the xanthan gum production-related gene as described in claim 2 into the genome of Xanthomonas oryzae.
[0011] As a preferred embodiment, the nucleic acid molecule is introduced into the genome of Xanthomonas oryzae via the pSRK-Km plasmid.
[0012] The present invention also provides a method for producing xanthan gum, comprising the following steps: fermenting and culturing the engineered bacteria for producing xanthan gum as described in claim 5 to obtain a fermentation product, and obtaining xanthan gum from the fermentation product.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] This invention provides the xanthan gum production-related gene XC_2876, which can be used for the genetic modification of xanthan gum-producing strains to increase xanthan gum production. The engineered strain obtained using this gene modification showed a significantly higher xanthan gum yield compared to Xanthomonas brassicae Xcc 8004. Detailed Implementation
[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0016] The materials used in the following embodiments include:
[0017] wild strain Xcc Plasmid 8004, pSRK-Km, originated from the preservation center of Guangdong Food and Drug Vocational College.
[0018] Restriction endonucleases Nde I and Hindi d III, T4 DNA ligase and other reagents were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.
[0019] The high-fidelity PCR mix was purchased from Guangzhou Qingke Biotechnology Co., Ltd.
[0020] PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0021] Example 1: Expression plasmid pSRK- XC_2876 Construction
[0022] according to XC_2876 Primers P1 and P2 were designed based on the gene sequence (SEQ ID NO.2) (underlined portions indicate restriction enzyme sites):
[0023] P1: 5'-AATTATG CATATG AGTCACCCATTTACGAG-3' (SEQ ID NO.3);
[0024] P2: 5'-AATT AAGCTT GGTTATTCCCTGGCGC-3' (SEQ ID NO. 4).
[0025] wild strain Xc of Xanthomonas brassicae c Using total DNA from 8004 as a template, and with primers P1 / P2 (pre-denaturation at 95℃ for 10 min, denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, and extension at 72℃ for 30 s), complete DNA was amplified. XC_2876 Gene sequence. Amplification system (50 μL): 1 μL total DNA, 1 μL each of P1 / P2 primers, 25 μL 2× high-fidelity PCR mix, and double-distilled water to 50 μL. Amplification conditions: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 50 s, 56℃ annealing for 30 s, and 72℃ extension for 1.5 min. Recovered DNA... XC_2876 Gene fragments Nde I and Hindi After digestion with dIII enzyme, the plasmid was ligated into the plasmid pSRK-Km. Sequencing verification yielded the expression plasmid pSRK-. XC_2876 .
[0026] Example 2 Engineered Strains Xcc 8004 / pSRK- XC_2876 Construction
[0027] Preparation of Escherichia coli using CaCl2 induction method E. coli S17-1 competent cells, 3 μL of plasmid pSRK- XC_2876 Add 100 μL E. coli After S17-1 competent cells were placed in an ice bath for 30 min, then heat-shocked at 42℃ for 120 s, 1 mL of antibiotic-free LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) was added, and the cells were incubated at 37℃ for 1 h. The cells were then plated on LB agar plates containing kanamycin (Km, 30 μg / ml) to obtain transformants. E. coli S17-1 / pSRK- XC_2876 .
[0028] Plasmid pSRK- was transferred using a conjugation transfer method. XC_2876 Import wild strains Xcc 8004, specific operating steps: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] E. coli S17-1 / pSRK- XC_2876 The recipient bacteria were cultured overnight at 37°C with shaking in 5 ml of liquid LB medium. Xcc 8004 was cultured overnight at 30°C with shaking in NYG liquid medium (5 g / L peptone, 3 g / L yeast extract, 20 g / L glycerol). The two bacterial cells were collected by centrifugation and mixed with 1 mL of NYG liquid medium. After washing twice, the mixture was resuspended in 0.1 mL of NYG, and the suspension was dropped onto antibiotic-free NYG solid medium. The culture was then air-dried and incubated upright for 48 h. The bacterial growth was washed with sterile water, diluted to a certain concentration, and spread onto 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 the engineered strain. Xcc 8004 / pSRK- XC_2876 .
[0029] Example 3: Detection of xanthan gum yield from different strains
[0030] The xanthan gum yield of different strains was accurately determined using the shake-flask fermentation method. Specific operational steps included: [The text abruptly shifts to a different topic] Xcc 8004 / pSRK- XC_2876 and wild strains Xcc Xanthan gum was precipitated by the 8004 strain in NYG liquid medium supplemented with 4 wt% glucose at 30°C for 4 days using a shaker. Four volumes of anhydrous ethanol were used to precipitate xanthan gum, which was then dried and weighed. The xanthan gum yield is shown in Table 1. Xcc 8004 / pSRK- XC_2876 The engineered strains showed a significant increase in xanthan gum production compared to the wild-type strains.
[0031] Table 1
[0032]
[0033] Finally, 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 the scope of protection of the present invention. 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 essence and scope of the technical solutions of the present invention.
Claims
1. The application of a xanthan gum yield-related gene in xanthan gum production, characterized in that, The target gene was cloned into the pSRK-Km vector to construct the recombinant expression plasmid pSRK-XC_2876. The recombinant expression plasmid pSRK-XC_2876 was introduced into Xanthomonas oryzae Xcc 8004 via conjugation transfer to obtain the engineered strain Xcc 8004 / pSRK-XC_2876. The amino acid sequence of the protein encoded by the target gene is shown in SEQ ID NO.
1.
2. The application of the xanthan gum production-related gene as described in claim 1 in the construction of xanthan gum-producing engineered bacteria, characterized in that, The target gene was cloned into the pSRK-Km vector to construct the recombinant expression plasmid pSRK-XC_2876. The recombinant expression plasmid pSRK-XC_2876 was introduced into Xanthomonas oryzae Xcc 8004 via conjugation transfer to obtain the engineered strain Xcc 8004 / pSRK-XC_2876. The amino acid sequence of the protein encoded by the target gene is shown in SEQ ID NO.
1.
3. An engineered bacterium that produces xanthan gum, characterized in that, The engineered bacteria were obtained by cloning the target gene into the pSRK-Km vector, constructing the recombinant expression plasmid pSRK-XC_2876, and introducing the recombinant expression plasmid pSRK-XC_2876 into Xanthomonas oryzae Xcc 8004 via conjugation transfer to obtain the engineered strain Xcc 8004 / pSRK-XC_2876. The amino acid sequence of the protein encoded by the target gene is shown in SEQ ID NO.
1.
4. The method for constructing the engineered bacteria that produce xanthan gum according to claim 3, characterized in that, The construction method includes the following steps: cloning the target gene into the pSRK-Km vector, constructing the recombinant expression plasmid pSRK-XC_2876, and introducing the recombinant expression plasmid pSRK-XC_2876 into Xanthomonas oryzae Xcc 8004 via conjugation transfer to obtain the engineered strain Xcc 8004 / pSRK-XC_2876; The amino acid sequence of the protein encoded by the target gene is shown in SEQ ID NO.
1.
5. A method for producing xanthan gum, characterized in that, The method includes the following steps: fermenting and culturing the engineered bacteria that produce xanthan gum as described in claim 3 to obtain a fermentation product, and then obtaining xanthan gum from the fermentation product.